Regulatory b cells and their uses

ABSTRACT

The present invention relates to a distinct B cell subset, B10 cells, that regulate T cell mediated inflammatory responses through the secretion of interleukin-10 (IL-10). The invention also relates to the use of B10 cells in the manipulation of immune and inflammatory responses, and in the treatment of disease. Therapeutic approaches involving adoptive transfer of B10 cells, or expansion of their endogenous levels for controlling autoimmune or inflammatory diseases and conditions are described. Ablation of B10 cells, or inhibition of their IL-10 production can be used to upregulate immunodeficient conditions, ameliorate infectious diseases and/or to treat tumors/cancer. Diagnostic applications are also encompassed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of U.S.Provisional Patent Application No. 61/370,677, filed Aug. 4, 2011, whichis incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States government support awarded bythe National Institutes of Health grant number National Cancer Institutegrant numbers CA 105001, CA 96547, and National Institute of Allergy andInfectious Disease grant number AI 56363. The United States may havecertain rights in this invention.

SEQUENCE LISTING

A Sequence Listing accompanies this application and is incorporatedherein by reference in its entirety. The Sequence Listing was filed withthe application as a text file on Aug. 4, 2011.

1. Introduction

The present invention relates to a distinct B cell subset, B10 cells,that regulate T cell mediated inflammatory responses through thesecretion of interleukin-10 (IL-10). The invention also relates to theuse of B10 cells in the manipulation of immune and inflammatoryresponses, and in the treatment of disease. Therapeutic approachesinvolving adoptive transfer of B10 cells, or expansion of theirendogenous levels for controlling autoimmune or inflammatory diseasesand conditions are described. Ablation of B10 cells, or inhibition oftheir IL-10 production can be used to upregulate immunodeficientconditions, and/or to treat tumors/cancer. Diagnostic applications arealso encompassed.

2. Background

The immune response can loosely be divided into two components: thehumoral immune response which involves antibody formation, and thecell-mediated immune response which involves the activation ofmacrophages, natural killer (NK) cells, antigen-specific cytotoxicT-lymphocytes, and the release of various cytokines in response toantigen. Typically, B lymphocytes (B cells) are characterized by theirrole in antibody production; whereas T lymphocytes (T cells) arecharacterized by their role in cell-mediated immunity. However, B cellspossess additional immune functions, including the production ofcytokines, and the ability to function as antigen presenting cells(APCs).

Once generated, immune responses need to be regulated to prevent theresponding effector cells from causing harmful effects. Immunoregulationhas traditionally been thought of as a function of T cells. Functionallydistinct regulatory T cell subsets have been identified and clearlydefined. For example, helper T cells that up-regulate the immuneresponse include T helper type 1 (Th1) cells that regulate cell-mediatedimmune responses, and T helper type 2 (Th2) cells that regulate thehumoral immune response. Suppressor T cells crucial for the maintenanceof immunological tolerance, currently referred to as T regulatory cells,include IL-10-producing T regulatory 1 (Tr1) cells, and TGF-β1-producingT helper type 3 (Th3) cells. Recent studies of autoimmune conditionsgave rise to the notion that B cells may also participate inimmunoregulation. However, regulatory B cell subsets have not beenclearly defined.

Multiple roles for B cells have been implicated in autoimmune diseases.B cells, a major immune cell population, can play a pathogenic role inacquired immune responses by producing autoantibodies that drive thedevelopment of autoimmune diseases. Certain therapies developed fortreating autoimmunity are aimed at depleting pathogenic B cells.However, the tools currently available are not specific for thepathogenic B cells and deplete most B cells. For example, B celldepletion in humans using CD20 monoclonal antibody (mAb) can beeffective in treating patients with various autoimmune disorders, suchas rheumatoid arthritis and lupus (Edwards et al., 2001, Rheumatol.40:205-11; Edwards et al., 2005, Rheumatol. 44:151-56; El Tal et al.,2006, J. Am. Acad. Dermatol. 55:449-59; Anolik et al., 2004, Arth.Rheum. 50:3580-90; Stasi et al., 2007, Blood 110:2924-30). CD20 is a Bcell-specific marker that is first expressed on the cell surface duringthe pre-B to immature B cell transition, but is lost upon plasma celldifferentiation (Tedder & Engel, 1994, Immunol. Today 15:450-54; Uchidaet al., 2004, Int. Immunol. 16:119-29). A recent phase II trial usingCD20 antibodies indicates clinical efficacy in multiple sclerosis (MS)patients (Hauser et al., 2008, N. Engl. J. Med. 358:676-88). However,the mechanisms underlying the effect of B cell depletion on diseaseactivity remains unknown. On the flip side, B cell depletion mayexacerbate disease. Indeed, B cell depletion was recently found toexacerbate ulcerative colitis in human clinical trials (Goetz et al.,2007, Inflamm Bowel Dis. 13:1365-8) and may contribute to thedevelopment of psoriasis (Dass et al., 2007, Arthritis Rheum.56:2715-8).

Over a decade ago, Janeway and colleagues (Wolf et al., 1996, J. Exp.Med. 184: 2271-2278) described studies designed to assess the role of Bcells in the course of autoimmune disease by inducing acute experimentalautoimmune encephalomyelitis (EAE) in B cell-deficient mice. EAE is anautoimmune disease of the central nervous system (CNS) that models humanmultiple sclerosis. Results showed that elimination of B cells did notprevent induction of autoimmunity. Instead, the lack of B cells seemedto exacerbate disease outcome, in that the B cell deficient mice did notfully recover as compared to wild-type mice. Thus, while B cells supplythe autoantibodies thought to be responsible for disease, theseinvestigators concluded that B cells are not required for activation ofdisease, and instead, that their presence is required to enhancerecovery. More recently, it was reported that B cell IL-10 productioncorrelated with recovery from EAE, a Th1-mediated autoimmune disease(Fillatreau et al., 2002, Nature Immunol. 3: 944-950). IL-10 is animmunoregulatory cytokine produced by many cell populations. IL-10 hasbeen shown to suppress cell-mediated immune and inflammatory responses.

Other recent studies in mouse models indicate that B cells and IL-10play a protective role in T cell-mediated inflammation, e.g., inTh2-mediated inflammatory bowel disease (Mizoguchi et al., 2002,Immunity 16:216-219), and in contact hypersensitivity (CHS) responses —acutaneous inflammatory immune reaction that is mediated by T cells insensitized individuals following subsequent contact with the sensitizingantigen (Enk, 1997, Mol. Med. Today 3:423-8). In particular, mice with Bcells deficient for CD19 expression (CD19^(−/−)) have augmented CHSresponses (Watanabe et al., 2007, Am. J. Pathol. 171:560-70). IL-1U mustbe involved in protection since neutralizing IL-10 through mAb treatmentenhances CHS responses, while systemic IL-10 administration reduces CHSresponses (Ferguson et al., 1994, J. Exp. Med. 179:1597-1604; Schwarz etal., 1994, J. Invest. Dermatol. 103:211-16).

On the basis of these and other studies, it has been proposed that, liketheir T cell counterparts, B cells can be divided into functionallydistinct regulatory subsets capable of inhibiting inflammatory responsesand inducing immune tolerance by mechanisms that include IL-10 and TGF-βproduction, secondary antigen presentation, and interaction with otherimmune cells either directly or through secreted antibodies. (Forreviews on the subject, see Mauri & Ehrenstein, 2007, TRENDS in Immunol.29: 34-40; and Mizoguchi & Bhan, 2006, J. Immunol. 176:705-710).

However, it remains unclear whether regulatory B cells represent aunique regulatory lineage tasked with maintaining self-tolerance the waythat naturally occurring regulatory T cells are. The generation ofregulatory B cells has been reported in multiple mouse models of chronicinflammation, although their existence in normal mice remains unknown(Mizoguchi & Bhan, 2006, J. Immunol. 176:705-10). Despite theidentification of a regulatory B cell subset generated in these mousemodels, no definitive murine phenotype has been established and, infact, only a general list of cell-surface markers associated withregulatory B cells exists (Mauri & Ehrenstein, 2007, Trends Immun.29:34-40). Furthermore, the existence of regulatory B cells in humansremains a matter of speculation, and the potential phenotypic markersfor human regulatory B cells are unknown (Mauri & Ehrenstein, 2007,Trends Immun. 29:34-40). A role for CD40 in the generation of regulatoryB cells and the induction of IL-10 production by these cells has beenpostulated (Inoue et al., 2006 Cancer Res. 66:7741-7747). Nonetheless,it has yet to be proven whether CD40 can be directly targeted, i.e.,with CD40 antibodies, as a means to generate regulatory B cells in vivo(Mauri & Ehrenstein, 2007, Trends Immun. 29:34-40).

Further complicating these issues, during immune responses, IL-10 isalso secreted by multiple cell types, including T cells, monocytes,macrophages, mast cells, eosinophils, and keratinocytes, and cansuppress both Th1 and Th2 polarization and inhibit antigen presentationand proinflammatory cytokine production by monocytes and macrophages(Asadullah et al., 2003, Pharmacol. Rev. 55:241-69). Clearly, it isunknown whether multiple B cell populations or a novel B cell subsetregulates inflammatory responses, whether regulatory B cells produceIL-10 or other cytokines directly, whether regulatory B cells havepotent activities in vivo, whether humans possess regulatory B cells,how regulatory B cells can be activated and/or expanded, and the role ofregulatory B cells in disease. To advance therapeutic application,subsets of immunoregulatory B cells need to be better defined and theirphenotype will need to be more closely correlated with their function invivo.

3. Summary

The present invention relates to a distinct regulatory B cell subset,termed B10 cells, that regulate T cell-mediated inflammatory and immuneresponses through secretion of IL-10. The invention also relates toharnessing this B10 cell subset for the manipulation of immune andinflammatory responses in humans and other mammals. Treatments fordiseases associated with diminished IL-10 levels, such as inflammatoryand autoimmune diseases are described, as well as treatments fordiseases associated with elevated IL-10 levels, such asimmunosuppression, infectious diseases and cancer.

Cellular compositions enriched for B10 cells, and methods for theirpreparation are described. B10 cells are characterized by increasingproduction of IL-10 after stimulation with a CD40 agonist or a TLR(Toll-like Receptor) agonist. For example, without being bound by theoryand without limitation, cellular compositions enriched by selectionusing both CD1d^(high) and CD5 as cellular markers will contain a higherpercentage of B10 cells than a population enriched using only one ofthese markers. Additionally, cellular compositions enriched by selectionusing both CD24^(high) and CD27 as cellular markers will contain ahigher percentage of B10 cells than a population enriched using only oneof these markers. Other markers that can be used in selection of B10cells include, without limitation, CD19, CD20, CD21, CD23, CD25, CD38,CD48, and CD148. These cellular compositions can be expanded and used totreat inflammatory and/or autoimmune conditions or diseases by adoptivetransfer.

In an alternative approach, therapeutic regimens designed to expand theendogenous population of B10 cells in subjects in need of such treatmentcan be used to treat inflammatory and/or autoimmune conditions ordiseases. This approach includes methods of treating a disease orcondition associated with diminished or insufficient levels of IL-10 ora condition or disease ameliorated by increasing levels of IL-10 or adisease or condition associated with inflammation or immunehyperresponsiveness by administering to a subject in need of suchtreatment a therapeutically effective amount of an agent that stimulatesthe expansion of IL-10 producing B10 cells or an agent that increasesproduction of IL-10 by B cells in the subject. The agent may be a TLRagonist or a CD40 agonist. In an alternative approach, the agent may beantibodies that activate and/or stimulate expansion of B10 cells, orincrease their production of IL-10.

In an alternative embodiment, methods of treating a disease or conditionassociated with elevated levels of IL-10 or insufficient or ineffectiveimmune responsiveness by administering to a subject in need of suchtreatment a therapeutically effective amount of an agent that kills,abrogates, or inhibits the function, localization, or expansion of B10cells or an agent that inhibits production of IL-10 by B10 cells in thesubject. The methods are suitable for treating diseases and/orconditions involving immunosuppression, infectious diseases or cancer bydepleting or ablating B10 cells in subjects in need thereof. In thisapproach, the agent may be antibodies that kill B10 cells, or inhibittheir function, proliferation or production of IL-10. In particular, theagent may include antibodies that induce homotypic adhesion and agentsthat selectively deplete or target B10 cells as opposed to other typesof B cells such as follicular B cells.

In yet another embodiment, methods for identifying B10 cells in patientsand/or patient samples are described for diagnosing the immune status ofaffected individuals. The methods include assaying for cells producingIL-10 or capable of producing IL-10 when treated with a CD40 agonist ora TLR agonist. Methods for assessing the number of B10 and B10pro cellsare also encompassed.

In still another aspect, methods of generating an antibody thatpreferentially or selectively depletes B10 cells is described. Themethod includes selecting an antibody that binds to a marker that isexpressed by B10 cells, assaying the antibody for the ability to inducehomotypic adhesion of B cells, and assaying the antibody for its abilityto deplete the B10 cell population. In some embodiments, the Fc portionof the antibody may be modified so that the mechanism of B10 celldepletion by the antibody is independent of the antibody's Fc region.The antibody may be selected for its ability to deplete the B10 cells bya method that is independent of antibody-dependent cellular cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC) and apoptosis.

In a further aspect, methods of selecting B10 cells is provided. Themethod includes selecting B lymphocytes in a sample from a subject,stimulating the B cells in vitro with PMA (phorbol 12-myristate13-acetate) and ionomycin for five hours and selecting IL-10 producingcells. The cells may also be selected by stimulating them for at least24 hours with a CD40 agonist or a TLR agonist prior to the addition ofPMA and ionomycin.

In a still further aspect, methods of inducing an IgG antibody responseto an antigen are described. The methods include administering theantigen to a subject and administering an agent that kills or inhibitsthe function, localization or expansion of B10 cells or inhibitsproduction of IL-10 by B10 cells to the subject. The administration ofthe agent with the antigen increases the antigen specific IgG antibodyproduction in the subject as compared to a subject administered theantigen alone. The antigen and the agent may be administered together,but need not be.

The invention is based, in part, on the identification of a rare B10cell subset that controls T cell-mediated immune and inflammatoryresponses in vivo. The principles of the invention are illustrated inanimal models in the studies described in the examples, infra, andresolve previously unexplained contradictions reported in the literaturefor the role of B cells in disease models such as EAE, arthritis, andinflammatory bowel disease. The examples described infra demonstrate:

-   -   a phenotypically unique B cell subset, B10 cells, with potent        regulatory activities in vivo;    -   a reliable method of intracellular cytokine staining that        clearly identifies B10 cells;    -   adoptive transfer of B10 cells has potent IL-10-dependent        regulatory functions during inflammation in vivo, which can        apply to other T cell-mediated inflammatory or autoimmune        diseases;    -   expansion of B10 cells in human CD19 transgenic mice results in        a decreased inflammatory response;    -   the absence of B10 cells in CD19-deficient mice results in        augmented T cell-mediated inflammation; and    -   the presence of B10 cells in healthy wild type mice (1-2% of        spleen B cells) and expansion of the population during contact        hypersensitivity responses.

4. DESCRIPTION OF THE FIGURES

FIG. 1 . CD22 mAb depletes B10 cells and increases IgG responses. Eightweek-old C57BL/6 mice were treated with CD22 mAb (MB22-10; 250 μg/mouse)or control mAb (B1; 250 μg/mouse) 7 days before analysis. (A)Representative CD1d and CD5 expression by CD19+B cells. Splenocytes werestained with CD1d, CD5, and CD19 mAbs with flow cytometry analysis ofviable cells. Results represent one mouse indicating the frequency ofCD1d^(hi)CD5⁺ B cells among total B cells within the indicated gates.Bar graphs indicate mean (±SEM) percentages and numbers of CD1d^(hi)CD5⁺B cells in one of two independent experiments with three mice in eachgroup. (B) IL-10 production by B cells. Splenocytes were cultured withLPS (10 μg/ml), PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (2μM) for 5 h, then stained with B220 and CD19 mAb to identify B cells,permeabilized, and stained using IL-10 mAb with flow cytometry analysisof viable cells. Representative results demonstrate the frequency ofIL-10-producing cells among total B220+ B cells within the indicatedgates. Bar graphs indicate mean (±SEM) percentages and numbers of Bcells that produced IL-10 in one of two independent experiments withthree mice in each group. Leukocytes from IL-10^(−/−) mice served asnegative controls to demonstrate specificity and to establish backgroundIL-10 staining levels. (C-D) B10 cells regulate IgG Ab responses.Wild-type mice were given CD22 or control mAb (n=3 per group) on day 0,and immunized with DNP-KLH without adjuvant on days 0 and 21. (C) SerumDNP-specific Abs were quantified by ELISA. (D) The frequency of B cellssecreting DNP-specific IgG was determined by ELISPOT analysis of spleencells harvested on day 28. (A-D) Significant differences between samplemeans are indicated: *, p<0.05; **, p<0.01.

FIG. 2 . Identification of human blood IL-10-competent B10 and B10procells. (A) Frequencies of mouse blood B10 cells after stimulation withPIM, LPS+PIM, or CpG+PIM for 5 h as described (see Yanaba et al., 2008,Immunity 28:639-650; Matsushita et al., 2008, J Clin. Invest.118:3420-3430; and Yanaba et al., 2009, J. Immunol. 182:7459-7472).Representative flow cytometry histograms are shown for one mouse withvalues from 5 mice shown in the scatter graph. (B) BFA treatment wasoptimal for visualizing human blood B10 cell numbers. Purified bloodmononuclear cells were cultured for 5 h with L+PIB or LPS+PIM as in (A)before immunofluorescence staining and flow cytometry analysis. Bargraph values represent mean (f SEM) B10 cell frequencies from 3individuals. (C) Representative gating strategy for identifyingcytoplasmic IL-10⁺ human B cells by flow cytometry. Blood mononuclearcells were cultured with LPS+PIB for 5 h and stained forimmunofluorescence analysis of viability and cell surface CD19expression. After membrane permeabilization, the cells were stained withIL-10 mAb. For flow cytometry analysis, single cells were identified bysinglet gating using forward scatter area (FSC-A) versus height (FSC-H)plots. The cells outside or below the indicated gate (left panel) wereexcluded cell doublets. The predominant lymphocyte population within thesingle cell gate was identified by forward (FSC) and side (SSC-A) lightscatter properties. Within the single cell lymphocyte population, thedead cells that were positive for Live/Dead staining were excluded fromthe analysis. The initial gate for identifying B cells was cell surfaceCD19 expression. Representative cytoplasmic IL-10 staining by viable,single CD19⁺ B cells is shown in the dot-plot histograms (right panels).Percentages indicate the frequencies of cytoplasmic IL-10⁺ B cellswithin the indicated gates among total CD19⁺ B cells. Blood mononuclearcells that were cultured with BFA alone before immunofluorescencestaining served as negative controls, with background staining similarto that obtained using isotype-matched control mAbs. (D) RepresentativeIL-10 production by human blood B cells from a normal individual withrelatively high B10 cell frequencies. B10 cells were identified bycytoplasmic IL-10 expression after in vitro culture with LPS, CpG,and/or PIB as indicated for 5 h. Blood mononuclear cells cultured withBFA alone served as negative controls for background IL-10 staining.Alternatively, B10 cell frequencies were examined after in vitro B10procell maturation by stimulation with LPS, CD40L+LPS, CpG, or CD40L+CpG,with PIB added during the final 5 h of 48 h cultures. As negativecontrols for IL-10 staining, the cells were also stimulated withCD40L+LPS or CD40L+CpG for 48 h, with only BFA added during the final 5h of culture. Percentages indicate the frequencies of cytoplasmic IL-10⁺B cells within the indicated gates among total CD19+ B cells. (E)Frequencies of blood B10 cells in individuals after stimulation with TLRagonists. Cell stimulation and analysis was as described in (C-D). Dotsrepresent results from single individuals after 5 h culture with BFAalone, PIB, or the indicated TLR agonist+PIB. Horizontal bars indicatemeans. (F) CD40L was optimal for inducing B10+B10pro cell maturationduring 48 h in vitro cultures. Purified blood mononuclear cells werecultured with either recombinant CD40L or CD40 mAb, plus LPS for 48 h.PIB was added during the final 5 h of culture. Bar graph valuesrepresent mean (±SEM) from 5 different individuals. Similar results wereobtained in 2 independent experiments. (G) Representative human bloodB10+B10pro cell frequencies after in vitro maturation and stimulation.Blood mononuclear cells were cultured for 48 h with media alone or mediacontaining CD40L, along with the indicated TLR agonists, with PIB addedduring the last 5 h of each culture. (A, E-G) Significant differencesbetween means of media controls and individual stimuli are indicated:*p<0.05, **p<0.01.

FIG. 3 . Human B10 and B10pro cells in cord blood, spleen and tonsil.(A) B10 and B10pro cells in human newborn blood. Mononuclear cells werecultured with the indicated stimuli with PIB added during the last 5 hof culture. Results from a representative cord blood sample are shownalong with graphs indicating IL-10⁺ B cell frequencies in individualnewborns. (B) Representative cytoplasmic IL-10 expression by humantissue B cells. B10 and B10pro cells were identified byactivation-induced cytoplasmic IL-10 expression as described in FIG. 2C.Cells cultured with BFA alone served as negative controls for backgroundIL-10 staining. (C) Human B10 cell frequencies determined after 5 or 48h in vitro cultures as described in FIG. 2 . Mouse spleen B10 and B10procell frequencies are shown for comparison. Dots represent results fromsingle individuals or mice. Significant differences between means of BFAor monensin (Mone) controls and individual stimuli are indicated:*p<0.05, **p<0.01.

FIG. 4 . Human B cell stimulation induces IL-10 transcription andsecretion in vitro. (A) Time course of II10 transcript induction.Purified blood CD19⁺ B cells were cultured with media alone (open bars)or CD40L plus CpG (filled bars) for the times indicated, with II10transcripts quantified by real-time RT-PCR analysis. Bar graphs indicatemean IL-10 (±SEM) concentrations of six different individuals.Significant differences between media alone and stimulated B cells areindicated: *p<0.05. Similar results were obtained in 2 independentexperiments. (B) II10 transcript expression correlates with IL-10secretion. Purified blood B cells were cultured with PMA and ionomycinfor 4 h before CD19 staining and secreted IL-10 capture (left panel).Cell surface IL-10⁺ and IL-10⁻ B cells were isolated using the indicatedgates and subsequently reassessed for IL-10 secretion (right panels)before relative II10 transcript levels were quantified by real-timeRT-PCR analysis. Mean fold-differences (±SEM) for 110 transcript levelsfrom 3 different individuals are shown, with transcript levelsnormalized so that the relative mean IL-10⁻ B cell value is 1.0.Significant differences between means are indicated: *p<0.05. Similarresults were obtained in 2 independent experiments. (C) Cell surfacesignals that regulate cytoplasmic IL-10 expression. Blood B cells werecultured with CpG, CD40L, and anti-IgM Ab (IgM) as indicated for 48 hwith PIB added during the final 5 h of culture. The B cells were thenstained with CD19 mAb, permeabilized, and stained using IL-10 mAb withflow cytometry analysis. Representative frequencies of IL-10-producingcells within the indicated gates are shown among total CD19⁺ B cells.Bar graphs indicate mean (±SEM) percentages of B cells that producedIL-10 in 5 different individuals. Similar results were obtained in 2independent experiments. (D) TLR agonists that induce B10 cell IL-10secretion. Purified blood CD19⁺ B cells were cultured with media alone,CD40L, or with TLR agonists and CD40L as indicated for 48 (open bars) or72 (filled bars) h. IL-10 secreted into the culture supernatant fluidwas quantified by ELISA. Bar graphs indicate mean IL-10 (4SEM)concentrations from ≥4 different individuals. Similar results wereobtained in 2 independent experiments. (E) LPS and CpG induce mouseblood B10 cell IL-10 secretion. Purified blood B cells were culturedwith media alone or with TLR agonists as indicated for 24 (open bars) or72 (filled bars) h. IL-10 secreted into the culture supernatant fluidwas quantified by ELISA. Bar graphs indicate mean IL-10 (±SEM)concentrations from triplicate cultures that represent one of threeindependent experiments. (D-E) Significant differences between means ofcells cultured in media alone and TLR agonists are indicated: *p<0.05,**, p<0.01.

FIG. 5 . Representative phenotypes of human and mouse blood and tissueB10 and B10pro cells. (A) Cell surface phenotype of human blood B10cells. Enriched B cells were cultured with L+PIB for 5 h. (B) Cellsurface phenotype of human blood B10+B10pro cells after 48 h stimulationwith CD40L+LPS with PIB added during the final 5 h of culture. (E) Cellsurface phenotype of mouse blood B10 cells. Purified blood mononuclearcells were cultured with L+PIM for 5 h. (F) Cell surface phenotype ofmouse blood B10 plus B10pro cells after 48 h stimulation with CD40 mAbwith LPS, PMA, ionomycin, and monensin added during the final 5 h ofculture. (C) Cell surface phenotype of human spleen B10 cells. PurifiedB cells were cultured with CpG+PIB for 5 h. (D) Cell surface phenotypeof human spleen B10+B10pro cells after 48 h stimulation with CD40L+CpGwith PIB added during the final 5 h of culture. (G) Cell surfacephenotype of mouse spleen B10 cells cultured with L+PIM for 5 h. (H)Cell surface phenotype of mouse spleen B10+B10pro cells after 48 hstimulation with CD40 mAb with L+PIM added during the final 5 h ofculture. (A-H) Cultured cells were stained for viability and cellsurface molecule expression, permeabilized, stained with anti-IL-10 mAb,and analyzed by flow cytometry. Representative cell surface moleculeexpression by IL-10⁺ (thick line) and IL10⁻ (thin line) CD19⁺ B cellsfrom three individuals or >3 mice is shown. Shaded histograms representisotype-matched control mAb staining.

FIG. 6 . Human blood B10 and B10pro cells are predominantlyCD24^(hi)CD27⁺. (A) Blood B10 cells were predominantlyCD24^(hi)CD27⁺CD48^(hi)CD148^(hi). Representative phenotypes of purifiedblood B cells cultured with CpG+PIB for 5 h before six-colorimmunofluorescence staining for viability, cell surface moleculeexpression, and cytoplasmic IL-10. Subsequently, CD24, CD27, CD48, andCD148 expression by IL-10⁺ (thick line) and IL-10⁻ (thin line) CD19⁺cells was assessed by flow cytometry. (B) Cell surface CD24, CD27, CD38,CD48, or CD148 expression were not affected during IL-10 induction.Representative phenotypes of CD19⁺ blood B cells cultured with media onice (thin line) or CpG+PIB (thick line) for 5 h beforeimmunofluorescence staining and flow cytometry analysis as in (A). (A-B)Shaded histograms represent isotype-matched control mAb staining.Results represent those obtained for 3 individuals. (C) Representativedistributions of B10 cells within B cell subsets of three individuals asdefined by CD24, CD27, IgD/CD38, and IgD/CD27 expression. Purified bloodB cells were cultured with LPS+PIB for 5 h before immunofluorescencestaining and flow cytometry analysis as in (A). The horizontal andvertical lines on each contour plot are shown for reference, with thelower left quadrants delineating the IgD⁻CD38⁻ and IgD⁻CD27⁻ subsets asdetermined by control mAb staining, respectively. Results representthose obtained for 5 individuals. (D) Blood B10 cells are predominantlyfound within the CD24^(hi)CD27⁺ B cell subset. Purified blood B cellswere cultured with LPS+PIB for 5 h before four-color immunofluorescencestaining for cell surface CD19, CD24, and CD27 expression andcytoplasmic IL-10 expression, with subsequent flow cytometry analysis.(E) B10pro cells derive from the CD24^(hi)CD27⁺ B cell subset. Purifiedblood B cells were stained for CD19, CD24, and CD27 expression andsorted into the CD24^(hi)CD27⁺ and CD24^(low)CD27⁺ B cell subsets asindicated by the gates shown. Each purified subset was reanalyzed byflow cytometry to determine purities, which were always >90% h.Subsequently, the purified B cells were cultured with CD40L plus eitherLPS or CpG for 48 h, with PIB added during the final 5 h of culture. Thecultured cells were then stained for cell surface CD19 and intracellularIL-10 expression with the relative percentages of IL-10⁺ B cells withinthe indicated gates determined. Similar results were obtained in 2independent experiments. (F) Clonal expansion of IL-10-producing B cellsafter CpG, but not LPS or CD40L stimulation in vitro. Blood mononuclearcells were labeled with CFSE and cultured with CD40L, and LPS or CPG for48-96 h, with PIB added for the last 5 h of culture. Histograms (right)represent CFSE expression by the IL-10⁺ (thick line) or IL-10⁻ (thinline) B cell subsets. Results are representative of two independentexperiments. (G) IL-10 is predominantly secreted by CD24^(hi)CD27⁺ Bcells. Purified blood B cells were sorted into the CD24^(hi)CD27⁺ andCD24^(low)CD27⁻ B cell subsets as in (E) and cultured with the indicatedstimuli for 72 h. IL-10 secreted into the culture supernatant fluid wasquantified by ELISA. Bar graphs indicate mean IL-10 (±SEM)concentrations from triplicate ELISA determinations. Significantdifferences between means from CD24^(hi)CD27⁺ and CD24^(low)CD27⁻ Bcells are indicated: **, p<0.01. Significant differences between meansfrom cells cultured in media or with stimuli are indicated: ^(##),p<0.01.

FIG. 7 . Blood B10 cell frequencies in patients with autoimmune disease.(A) Representative B cell cytoplasmic IL-10 expression by control (Ctrl)individuals, and SLE, RA, SjS, BD, and MS patients with relatively highB10 cell frequencies after in vitro CpG plus PIB stimulation for 5 h.B10+B10pro cell maturation was induced by 48 h CD40L plus CpGstimulation, with PIB added during the final 5 h of culture. Percentagesindicate cytoplasmic IL-10⁺ B cell frequencies within the indicatedgates among total CD19⁺ B cells. (B) IL-10⁺ B cell frequencies incontrol individuals and patients as represented in (A) with each dotrepresenting single individuals. Horizontal bars indicate group means,the solid horizontal lines indicate means+2 SD (95% confidence interval)for controls, while dashed lines represent means+2 SD for all values.The patients are described in Table 1. (C) Relationship between B10 andB10pro cell frequencies in control individuals and autoimmune patientsafter in vitro culture with LPS or CpG plus PIB for 5 h. B10⁺B10procells were identified by cytoplasmic IL-10 expression after 48 hstimulation with CD40L plus LPS or CpG with PIB added during the final 5h of culture. (D) Relative frequencies of B10 cells and B10⁺B10pro cellsidentified for control individuals and patients with autoimmune diseaseare compared after CpG or LPS stimulation as shown in (B) with each dotrepresenting a single individual. (E) Relationship between cytoplasmicIL-10 expression levels and B10pro cell frequencies in controlindividuals and patients. Each dot representing single individuals afterstimulation with CD40L plus CpG, with PIB added during the final 5 h of48 h cultures. Linear mean fluorescence intensities (MFI) for IL-10⁺ andIL-10⁻ B cells were determined using the gates indicated in (A) with thevalues shown representing a ratio of IL-10⁺ to IL-10⁻ MFIs. A linearregression line (±95% prediction bands, dashed lines) is shown forreference.

5. DETAILED DESCRIPTION

The present invention relates to a phenotypically distinct B cellsubset, B10 cells, that regulate T cell-mediated inflammatory and immuneresponses through secretion of IL-10. The invention also relates toharnessing B10 cells for the manipulation of the immune and inflammatoryresponses, and for the treatment of diseases, disorders and conditionsassociated with altered IL-10 levels, including inflammatory andautoimmune diseases, as well as immunosuppression, infectious diseasesand cancer in humans and other mammals.

Cellular compositions enriched for B10 cells, and methods for theirpreparation are described. The B10 cells are characterized by theability to produce IL-10, in particular when stimulated with a CD40 orTLR agonist. These cellular compositions can be expanded and used inadoptive transfer therapies to treat conditions associated withdiminished IL-10 production or those ameliorated by increased levels ofIL-10, e.g., inflammatory and/or autoimmune conditions or diseases. Inan alternative approach, therapeutic regimens designed to expand theendogenous population of B10 cells, or increase their production ofIL-10 can be used to treat a disease or condition associated withdiminished levels of IL-10 or ameliorated by increased levels of IL-10such as inflammatory, immune hyperresponsive and/or autoimmuneconditions or diseases in subjects in need thereof. In this approach,agents capable of activating or stimulating B10 cells are administeredto the subject in need of such treatment. The agent may be a TLR agonistor a CD40 agonist. In an alternative approach, the agent may beantibodies that activate and/or stimulate expansion of B10 cells, orincrease their production of IL-10. Expansion can be accomplished invivo (e.g., by direct administration of the agent such as an antibody orreceptor agonist) or ex vivo (e.g., by activating and/or expanding thecells obtained from the subject and returning the activated cells to thesubject).

Methods of treating a disease or condition associated with elevatedlevels of IL-10 or insufficient or ineffective immune responsiveness arealso provided. These methods include administering a therapeuticallyeffective amount of an agent that kills, abrogates, or inhibits thefunction, localization, or expansion of B10 cells or an agent thatinhibits production of IL-10 by B10 cells to a subject in need of suchtreatment. The methods are suitable for treating diseases and/orconditions involving immunosuppression, infectious diseases or cancer bydepleting or ablating B10 cells in subjects in need thereof. In thisapproach, the agent may be antibodies that kill B10 cells, or inhibittheir function, proliferation or production of IL-10. In particular, theagent may include antibodies that induce homotypic adhesion and agentsthat selectively deplete or target B10 cells as opposed to other typesof B cells such as follicular B cells.

Methods for identifying B10 cells in patients and/or patient samples aredescribed for diagnosing the immune status of affected individuals. Themethods include assaying for cells producing IL-10 or capable ofproducing IL-10 when treated with a CD40 agonist or a TLR agonist.Methods for assessing the number of B10 and B10pro cells in a subjectare also encompassed. The immune status of the individual may be usefulin predicting the likelihood of contracting a particular disease, suchas an autoimmune disease, or in predicting responsiveness to particulardiseases or particular therapeutics.

In another embodiment, a method for generating an antibody thatpreferentially or selectively depletes B10 cells as compared to othersubsets of B cells is provided. The method comprises: (i) selecting anantibody that binds to a marker that is expressed by B10 cells includingbut not limited to, e.g. CD1d, CD5, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD27, CD38, CD40, CD48, CD72, and CD148; (ii) assaying theantibody for the ability to induce homotypic adhesion of B cells (KansasG S, Wood G S, Tedder T F. Expression, distribution and biochemistry ofhuman CD39: Role in activation-associated homotypic adhesion oflymphocytes. J Immunol. 1991; 146:2235-2244; Kansas G S, Tedder T F.Transmembrane signals generated through MHC class II, CD19, CD20, CD39and CD40 antigens induce LFA-1-dependent and -independent adhesion inhuman B cells through a tyrosine kinase-dependent pathway. J Immunol.1991; 147: 4094-4102.;Wagner N, Engel P, Vega M, Tedder T F. Ligation ofMHC class I and class II molecules leads to heterologous desensitizationof signal transduction pathways that regulate homotypic adhesion inhuman lymphocytes. J Immunol. 1994; 152:5275-5287.); (iii) assaying theantibody for the ability to deplete the B10 cell population. Optionally,the ability of the antibody to deplete or avoid depletion of other Bcell subsets may also be assessed. In some embodiments, the Fc portionof the antibody may be modified so that the mechanism of B10 celldepletion by the antibody is independent of the antibody's Fc region.The antibody may be selected for its ability to deplete the B10 cells bya method that is independent of antibody-dependent cellular cytotoxicity(ADCC), complement-dependent cytotoxicity (CDC) and apoptosis.

Methods of selecting B10 cells are also provided. The method includesselecting B lymphocytes in a sample from a subject, stimulating the Bcells in vitro with PMA and ionomycin for five hours and selecting IL-10producing cells. The cells may also be selected by stimulating them forat least 24 hours with a CD40 agonist or a TLR agonist prior to theaddition of PMA and ionomycin. The cells may be further selected byscreening for markers of B10 cells including but not limited toexpression of CD1d, CD5, CD19, CD2, CD2, CD22, CD23, CD24, CD25, CD27,CD38, CD40, CD48, CD72, or CD148 or the relative levels of expression ofthese markers on the cell surface.

Methods of inducing an IgG antibody response to an antigen, such as in avaccine or vaccination protocol are described. The methods includeadministering the antigen to a subject and administering an agent thatkills or inhibits the function, localization or expansion of B10 cellsor inhibits production of IL-10 by B10 cells to the subject. Theadministration of the agent with the antigen increases the antigenspecific IgG antibody production in the subject as compared to a subjectadministered the antigen alone. The antigen and the agent may beadministered together, but need not be.

5.1 The B10 Cell Subset

The present invention relates to a regulatory subset of the normal Bcell population, B10 cells, with the ability to produce IL-10. Theinvention also relates to therapeutic uses of B10 cells.

The phenotype of B10 cells can be determined by antibody staining andflow cytometry, FACS, using antibodies to phenotypic markers of B10cells, and techniques known in the art, including but not limited tothose described in the examples, infra. See, e.g., Section 6 et seq andU.S. Patent Publication No. 2011/0135666. The invention is based, inpart, on the surprising discovery that cellular compositions enriched byselection for B10 cell cellular markers (such as CD24^(high)CD27⁺ orCD1d^(high)CD5⁺) will contain a high percentage of IL-10 producing Bcells. It is also based on the discovery that these B10 cells can beselected based on their ability to produce IL-10 when stimulated withCD40 agonists or TLR agonists.

The ability of the cells to produce IL-10 can be assessed by measuringIL-10 production in naïve cells and in cultured cells stimulated withLPS (lipopolysaccharide), PMA (phorbol 12-myristate 13-acetate),ionomycin, CpG oligodeoxynucleotides or comparable stimulatory Toll-likereceptor (TLR) agonists, or with an agonist of CD40 (e.g., using anantibody to CD40). Production of IL-10 by the cells can be assessed byassaying for IL-10 in the cell culture supernatant. In addition,production of IL-10 can be verified directly by intracellular cytokinestaining after additional treatment with Brefeldin A or monensin.Standard immunoassays known in the art can be used for such purpose.Examples of assays for IL-10 production are described in Section 7,infra. While IL-10 is produced at low levels in naïve B10 cells, IL-10production is increased in response to stimulation.

5.1.1 Cellular Compositions Enriched in B10 Cells

The enriched, isolated and/or purified B10 cell subset composition cancomprise anywhere from 0.5% to 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90° %, at least 95%, at least 99%, or 100% B10 cells (as determined,e.g., by the assays described above). In a preferred embodiment, theenriched/purified B10 cell subset comprises greater than 50% B10 cells.In a more preferred embodiment, the enriched/purified B10 cell subsetcomprises greater than 75% B10 cells. In a still more preferredembodiment, the enriched/purified B10 cell subset comprises greater than90% B10 cells. In certain embodiments, the enriched, isolated and/orpurified B10 cells have a CD1d^(high)CD5⁺ phenotype. In certainembodiments, the enriched, isolated and/or purified B10 cells have aCD24^(hi)CD27⁺ phenotype.

The enriched, isolated and/or purified B10 cells can be obtained from amammalian subject, including but not limited to rodents, e.g. mice,rats; livestock, e.g. pigs, horses, cows, etc., pets, e.g. dogs, cats;and primates, e.g. humans. In one embodiment, the subject is an animalmodel of an IL-10 associated disease.

Alternatively, B10 cells may be enriched/purified from any tissue wherethey reside including, but not limited to, blood (including bloodcollected by blood banks and cord blood), spleen, bone marrow, lymphnodes, tissues removed and/or exposed during surgical procedures, andtissues obtained via biopsy procedures. Tissues/organs from which theB10 cells are enriched, isolated, and/or purified may be isolated fromboth living and non-living subjects, wherein the non-living subjectsinclude organ donors.

Methods for the isolation of B10 cells are based on selecting cellshaving the B10 cell-specific markers. In a specific embodiment, the B10cell-specific markers comprise CD1d^(high) and CD5. In another specificembodiment, the B10 cell-specific markers comprise CD24^(high) and CD27.In certain embodiments, additional B cell-specific markers can be usedfor selection including, but not limited to, CD1d, CD19, CD20, CD21,CD23, CD24, CD25, CD38, CD48, and CD148. Several of these markers orcombinations of these markers can be used to specifically select B10cells. In a particular aspect of this embodiment, a B10 cells areenriched/purified by flow cytometry as demonstrated in the examplesdescribed in Section 6 and 7, infra. However, a variety of cellseparation techniques known in the art can be used, including but notlimited to magnetic separation using antibody-coated magnetic beadsand/or particles, FACS, affinity chromatography, affinity columnseparation, “panning” with antibody attached to a solid matrix, densitygradient centrifugation, and counter-flow centrifugal elutriation. (See,e.g., Kumar and Lykke, 1984, Pathology, 1:53-62).

According to these embodiments, a cellular composition enriched for B10cells that has been enriched by selection using both CD1d^(high) and CD5as cellular markers or CD24^(high) and CD27 as cellular markers willcontain a higher percentage of B10 cells than one enriched using onlyone of these markers. The use of these markers to isolate/enrich/purifyB10 cells has several advantages. Using these cell surface markers, asopposed to intracellular IL-10 as a marker, allows for theselection/sorting of the IL-10 producing B cell population withoutpermeabilizing the cells, which would make them therapeutically useless.Once enriched, the cells may be further purified or expanded bystimulating or activating the cells with a CD40 agonist or a TLRagonist. These agonists may be used in combination with PMA andionomycin.

B10 cells can also be isolated by negatively selecting against cellsthat are not B10 cells. This can be accomplished by performing a lineagedepletion, wherein cells are labeled with antibodies for particularlineages such as the T lineage, the macrophage/monocyte lineage, thedendritic cell lineage, the granulocyte lineages, the erythrocyteslineages, the megakaryocytes lineages, and the like. Cells labeled withone or more lineage specific antibodies can then be removed either byaffinity column processing (where the lineage marker positive cells areretained on the column), by affinity magnetic beads or particles (wherethe lineage marker positive cells are attracted to the separatingmagnet), by “panning” (where the lineage marker positive cells remainattached to the secondary antibody coated surface), or bycomplement-mediated lysis (where the lineage marker positive cells arelysed in the presence of complement by virtue of the antibodies bound totheir cell surface). Another lineage depletion strategy involvestetrameric complex formation. Cells are isolated using tetramericanti-human antibody complexes (e.g., complexes specific for multiplemarkers on multiple cell types that are not markers of B10 cells, givenin more detail infra) and magnetic colloid in conjunction with StemSepcolumns (Stem Cell Technologies, Vancouver, Canada). The cells can thenoptionally be subjected to centrifugation to separate cells havingtetrameric complexes bound thereto from all other cells.

In a certain embodiment, the enriched/purified B10 cells can be storedfor a future use. In this regard, the B10 cells can be stored by“cryopreservation.” Cryopreservation is a process where cells or wholetissues are preserved by cooling to low sub-zero temperatures, such as77 K or −196° C. in the presence of a cryoprotectant. At these lowtemperatures, any biological activity, including the biochemicalreactions that would lead to cell death, is effectively stopped. Storageby cryopreservation includes, but is not limited to, storage in liquidnitrogen, storage in freezers maintained at a constant temperature of 0°C., storage in freezers maintained at a constant temperature of −20° C.,storage in freezers maintained at a constant temperature of −80° C., andstorage in freezers maintained at a constant temperature of lower than−80° C. In one aspect of this embodiment, the cells may be“flash-frozen,” e.g., in ethanol/dry ice or in liquid nitrogen prior tostorage. In another aspect of this embodiment, the cells can bepreserved in medium comprising a cryprotectant including, but notlimited to dimethyl sulfoxide (DMSO), glycerol, ethylene glycol,propylene glycol, sucrose, and trehalose. Other methods of storingbiological matter are well known to those of skill in the art, such as“hibernation,” wherein cells are stored at temperatures above freezingor by preservation of the cells in a “static” state, as described inU.S. patent application publication No. 2007/0078113, incorporatedherein by reference in is entirety.

In certain embodiments, B10 cells can be obtained from a subject in needof therapy or suffering from a disease associated with elevated ordiminished levels of IL-10. Alternatively, B10 cells can be obtainedfrom a donor, preferably a histocompatibility matched donor. B10 cellsmay be harvested from the peripheral blood, bone marrow, spleen, or anyother organ/tissue in which B10 cells reside in said subject or donor.Ina further aspect, the B10 cells may be isolated from a pool ofsubjects and/or donors, or from pooled blood.

When the population of B10 cells is obtained from a donor distinct fromthe subject, the donor is preferably syngeneic, but can also beallogeneic, or even xenogeneic, provided the cells obtained aresubject-compatible in that they can be introduced into the subject.Allogeneic donor cells are preferably human-leukocyte-antigen(HLA)-compatible, and are typically administered in conjunction withimmunosuppressive therapy. To be rendered subject-compatible, xenogeneiccells may be subject to gamma irradiation or PEN110 treatment asdescribed (Fast et al., 2004, Transfusion 44:282-5).

5.1.2. Enrichment of B10 Cells

B10 cells can be enriched by selecting cells having the CD1d^(high)CD5⁺surface markers or the CD24^(high)CD27⁺ surface markers and separatingusing automated cell sorting such as fluorescence-activated cell sorting(FACS), solid-phase magnetic beads, etc. as demonstrated in examplesdescribed in sections 6 and 7 infra. To enhance enrichment, positiveselection may be combined with negative selection; i.e., by removingcells having surface markers specific to non-B cells and/or thosespecific to non-B10 cells. Non-limiting examples of methods of negativeselection are described supra. Exemplary surface markers specific tonon-B10 cells include CD3, CD4, CD7, CD8, CD15, CD16, CD34, CD56, CD57,CD64, CD94, CD116, CD134, CD157, CD163, CD208, F4/80, Gr-1, and TCR.

The cells may be used to make pharmaceutical compositions.Pharmaceutical compositions comprising the B10 cells described hereinand a pharmaceutically acceptable carrier are provided. Apharmaceutically acceptable carrier is any carrier suitable for in vivoadministration. Examples of pharmaceutically acceptable carrierssuitable for use in the composition include, but are not limited to,water, buffered solutions, glucose solutions, oil-based or bacterialculture fluids. Additional components of the compositions may suitablyinclude, for example, excipients such as stabilizers, preservatives,diluents, emulsifiers and lubricants. Examples of pharmaceuticallyacceptable carriers or diluents include stabilizers such ascarbohydrates (e.g., sorbitol, mannitol, starch, sucrose, glucose,dextran), proteins such as albumin or casein, protein-containing agentssuch as bovine serum or skimmed milk and buffers (e.g., phosphatebuffer). Especially when such stabilizers are added to the compositions,the composition is suitable for freeze-drying or spray-drying. Thecomposition may also be emulsified.

5.2 Expansion of the B10 Cell Subset and/or Enhancing their Productionof IL-10

In a particular embodiment, expansion of the B10 cell population isachieved by contacting a population of B10 cells with a stimulatorycomposition sufficient to cause an increase in the number of B10 cells.This may be accomplished by contacting the enriched, isolated and/orpurified B10 cell subset with a mitogen, cytokine, growth factor,antibody, CD40 agonist or TLR agonist. The B10 cells are preferablyexpanded at least 10-fold and preferably at least 50, 100, 200, 300,500, 800, 1000, 10,000, or 100,000-fold. In a specific aspect of thisembodiment, the expanded B10 cell population retains all of thegenotypic, phenotypic, and functional characteristics of the originalpopulation. In another embodiment, one or more of the characteristics ofthe B10 cell population is lost or modified following expansion.

Levels of IL-10 produced by the B10 cell subset can be increased by,e.g., administration of agonists to the B cell surface receptor CD40.Non-limiting examples of CD40 agonists include CD40 antibodies andfragments thereof, the CD40 ligand and polypeptide fragments thereof,small molecules, synthetic drugs, peptides (including cyclic peptides),polypeptides, proteins, nucleic acids, synthetic or natural inorganicmolecules, mimetic agents, and synthetic or natural organic molecules.

In a certain embodiment, the CD40 agonist is a CD40 antibody. The CD40antibodies of the invention can be of any form, as disclosed above.Antibodies to CD40 are known in the art (see, e.g., Buhtoiarov et al.,2005, J. Immunol. 174:6013-22; Francisco et al., 2000, Cancer Res.60:3225-31; Schwulst et al., 2006, 177:557-65, herein incorporated byreference in their entireties).

Levels of IL-10 produced by the B10 cell subset can also be increasedby, e.g., administration of agonists to TLRs on the B10 cell surface. Inparticular embodiments the TLR agonist may be an agonist of TLR1, TLR4,TLR6, TLR7, or TLR9. These agonists include natural ligands of thesereceptors and non-natural ligands or agonists. Non-limiting examples ofTLR agonists include LPS, CpG oligodeoxynucleotides, Pam3CSK4,Pam2CGDPKHPKSF, or Imiquimod and also include variations of thesemolecules or other small molecules, synthetic drugs, peptides (includingcyclic peptides), polypeptides, proteins, nucleic acids, synthetic ornatural inorganic molecules, mimetic agents, and synthetic or naturalorganic molecules.

Expansion of IL-10 production by the B10 cell subset can beadvantageously achieved ex vivo, e.g., by isolating the enriched B10cell population and contacting the cells with a CD40 agonist or a TLRagonist. In an aspect of this embodiment, the cells are contacted with aCD40 agonist or a TLR agonist and relevant antigen(s). In a specificaspect of this embodiment, the cells are contacted with a CD40 agonist,a TLR agonist and relevant antigen(s).

5.3 Ablation of the B10 Cell Subset and/or Inhibiting their Productionof IL-10

The B10 cell subset can be ablated by, e.g., engaging a B cell surfacemarkers e.g., CD20 or CD22. Non-limiting examples of compounds capableof engaging B cell surface markers to ablate the B10 cell populationinclude antibodies and fragments thereof, the ligand for the cellsurface marker and fragments thereof, ligand mimetics, small molecules,synthetic drugs, peptides (including cyclic peptides), polypeptides,proteins, nucleic acids, synthetic or natural inorganic molecules,mimetic agents, and synthetic or natural organic molecules. Antibodiesto B cell surface markers are known in the art (for CD22 see, e.g.,Tuscano et al., 2003, Blood 101:3641-7; US 2004/0001828 A1; and US2007/0264360, for CD20 see, e.g., US 2009/0136516 incorporated byreference herein in their entireties).

Alternatively, a bispecific antibody for CD1d and CD5 may be used totarget the B10 cell subset (these will be referred to herein asbispecific “CD1d/CD5” antibodies). Bispecific antibodies can be preparedfrom CD1d and CD5 antibodies using techniques that are known in the art(see, e.g., U.S. Pat. Nos. 5,534,254, 5,837,242, 6,492,123; U.S. Patentapplication publication Nos. 20040058400 and 20030162709, which are allherein incorporated by reference in their entireties). In anotherembodiment, a bispecific antibody for CD24 and CD27 may be used totarget the B10 cell subset.

In order to kill or ablate the B10 cell subset, targeting antibodies(e.g., CD20, CD22, bispecific CD1d/CD5, or bispecific CD24/CD27) of anisotype that mediate ADCC (antibody-dependent and mediated toxicity) orCDC (complement-dependent cytotoxicity) can be used. Of the varioushuman immunoglobulin classes, IgG1, IgG2, IgG3, IgG4 and IgM are knownto activate complement. Human IgG1 and IgG3 are known to mediate ADCC.Antibodies to CD20 may be used to deplete B10 cells selectively. Theantibodies demonstrated to target the IL-10 producing B10 cell subsetwere antibodies that were not capable of inducing ADCC, CDC orapoptosis. Instead the effective antibodies were those that inducedhomotypic adhesion. Thus, antibodies to B cell surface markers with IgG3or IgG2b Fc regions which do not efficiently engage most Fcγ receptors,but induce homotypic adhesion are useful in the methods describedherein. Optionally the Fc portion of a known antibody may be modified sothat the mechanism of depletion of B10 cells is independent of theantibody's Fc region.

Antibodies targeting the B10 cell subset can be further conjugated to acytotoxic agent, using methods known in the art (see, e.g., DiJoseph etal., 2004, Clin. Cancer Res. 10:8620-9). This may be preferred whenusing antibodies or antibody fragments that do not mediate ADCC or CDC.Non-limiting examples of cytotoxic agents include antimetabolites (e.g.,cytosine arabinoside, aminopterin, methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, and 5-fluorouracil decarbazine); alkylatingagents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan,carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiammine-platinum (II) (CDDP), and cisplatin); vincaalkaloid; anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); calicheamicin; CC-1065and derivatives thereof; auristatin molecules (e.g., auristatin PHE,bryostatin-1, and dolastatin-10; see Woyke et al., Antimicrob. AgentsChemother. 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001),Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad,et al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporatedby reference herein in their entireties); DNA-repair enzyme inhibitors(e.g., etoposide or topotecan); kinase inhibitors (e.g., compoundST1571, imatinib mesylate (Kantarjian et al., Clin. Cancer Res.8(7):2167-76 (2002)); demecolcine; and other cytotoxic agents (e.g.,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracenedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof and those compounds disclosed in U.S. Pat. Nos.6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459, all of which are incorporated byreference herein in their entirety); farnesyl transferase inhibitors(e.g., R115777, BMS-214662, and those disclosed by, for example, U.S.Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959,6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615,6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487,6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338,6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786,6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465,6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853,6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and6,040,305, all of which are herein incorporated by reference in theirentirety); topoisomerase inhibitors (e.g., camptothecin, irinotecan,SN-38, topotecan, 9-aminocamptothecin, GG211 (GI147211), DX-8951f,IST-622, rubitecan, pyrazoloacridine, XR5000, saintopin, UCE6, UCE1022,TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506,and rebeccamycin); bulgarein; DNA minor groove binders such as Hoechstdye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; antisense oligonucleotides (e.g.,those disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,5,734,033, and 5,618,709, all of which are herein incorporated byreference in their entirety); adenosine deaminase inhibitors (e.g.,fludarabine phosphate and 2-chlorodeoxyadenosine); and pharmaceuticallyacceptable salts, solvates, clathrates, and prodrugs thereof.

In another embodiment, the targeting antibodies, such as a CD20, CD22,bispecific CD1d/CD5, or bispecific CD24/CD27 antibody, can be conjugatedto a radioactive metal ion, such as the alpha-emitters²¹¹astatine,²¹²bismuth, ²¹³bismuth; the beta-emitters ¹³¹iodine, ⁰⁹yttrium,¹⁷⁷lutetium, ¹⁵³samarium, and ¹⁰⁹palladium; or macrocyclic chelatorsuseful for conjugating radiometal ions, including but not limited to,¹³¹indium, ¹³¹L, ¹³¹yttrium, ¹³¹3holmium, ¹³¹samarium, to polypeptidesor any of those listed supra. In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid(DOTA), which can be attached to the antibody via a linker molecule.Such linker molecules are commonly known in the art and described inDenardo, et al., 1998, Clin Cancer Res 4(10):2483-90; Peterson, et al.,1999, Bioconjug Chem 10(4):553-7; and Zimmerman, et al., 1999, Nucl MedBiol 26(8):943-50, each incorporated by reference herein in theirentireties.

In still another embodiment, the targeting antibody, e.g. the CD20,CD22, bispecific CD1d/CD5, or bispecific CD24/CD27 antibody isconjugated to a proteinaceous agent that modifies a given biologicalresponse and leads to cytotoxicity. In one embodiment, the antibody isconjugated to a plant-, fungus-, or bacteria-derived toxin. Non-limitingexamples of such toxins include A chain toxins, ribosome inactivatingproteins, ricin A, deglycosylated ricin A chain, abrin, alpha sarcin,aspergillin, restrictocin, ribonucleases, diphtheria toxin, bacterialendotoxin, saporin toxin, Granzyme B or the lipid A moiety of bacterialendotoxin, cholera toxin, or Pseudomonas exotoxin and derivatives andvariants thereof.

In another embodiment, an antagonist capable of engaging a B cellsurface marker such as CD20, CD22, CD1d or CD5 may be used to ablate theB10 cell population is a synthetic ligand targeted to a B10 cellspecific marker, such as that described in Collins et al., 2006, J.Immunol. 5:2994-3003, incorporated herein by reference in its entirety.In one aspect of this embodiment, the synthetic ligand may be furtherconjugated to a toxin, such as the saporin toxin.

Alternatively, a compound capable of engaging a marker or markers on theB10 cell subset can inhibit the production of IL-10 by the B10 cells.Non-limiting examples of such compounds include antibodies and fragmentsthereof, small molecules, synthetic drugs, peptides (including cyclicpeptides), polypeptides, proteins, nucleic acids, synthetic or naturalinorganic molecules, mimetic agents, and synthetic or natural organicmolecules. In one embodiment, the compound engages CD22. In an aspect ofthis embodiment, the compound is a CD22 antibody. In another aspect ofthis embodiment, the compound engages CD5. In an aspect of thisembodiment, the compound is a CD5 antibody. In another aspect of thisembodiment, the compound engages CD1d. In an aspect of this embodiment,the compound is a CD1d antibody. In still another aspect of thisembodiment, the compound is a bispecific CD1d/CD5 antibody. In anotheraspect of this embodiment, the compound engages CD24. In an aspect ofthis embodiment, the compound is a CD24 antibody. In another aspect ofthis embodiment, the compound engages CD27. In an aspect of thisembodiment, the compound is a CD27 antibody. In still another aspect ofthis embodiment, the compound is a bispecific a CD24/CD27 antibody. Inyet another aspect of this embodiment, the compound engages CD19. In anaspect of this embodiment, the compound is a CD19 antibody. In oneembodiment, the compound engages CD20. In an aspect of this embodiment,the compound is a CD20 antibody. Alternatively, the compound may bindone or more of CD1d, CD5, CD19, CD20, CD21, CD22, CD24, CD27, CD38,CD40, CD48, CD72, or CD148. In one aspect the compound may be anantibody or a bispecifica antibody directed to one or more of CD1d, CD5,CD19, CD20, CD21, CD22, CD24, CD27, CD38, CD40, CD48, CD72, or CD148.

5.4 Production of Therapeutic Antibodies

Antibodies that target, activate, inhibit and/or kill the B10 cellsubset and which can be used in the therapeutic regimens describedherein can be made using techniques well known in the art. The practiceof the invention employs, unless otherwise indicated, conventionaltechniques in molecular biology, microbiology, genetic analysis,recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotidesynthesis and modification, nucleic acid hybridization, and relatedfields within the skill of the art. These techniques are described inthe references cited herein and are fully explained in the literature.See, e.g., Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel etal, Current Protocols in Molecular Biology, John Wiley & Sons (1987 andannual updates); Current Protocols in Immunology, John Wiley & Sons(1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: APractical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotidesand Analogues: A Practical Approach, IRL Press; Birren et al (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press, each of which is incorporated by reference herein inits entirety.

Antibodies for use in the methods of the invention include, but are notlimited to, synthetic antibodies, monoclonal antibodies (mAbs),recombinantly produced antibodies, multispecific antibodies (includingbi-specific antibodies), human antibodies, humanized antibodies,chimeric antibodies, intrabodies, diabodies, single-chain Fvs (scFv)(e.g., including monospecific, bispecific, etc.), camelized antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above.

In particular, antibodies to be used in the methods of the inventioninclude immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that binds to a CD1d, CD5, CD22, CD24, CD27 or CD40antigen, or bispecifically to the CD1d and CD5 antigens or the CD24 andCD27 antigens. The immunoglobulin molecules can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass of immunoglobulin molecule.

Variants and derivatives of antibodies include antibody fragments thatretain the ability to specifically bind to an epitope. In certainembodiments, fragments include Fab fragments; Fab′; F(ab′)₂; abispecific Fab; a single chain Fab chain comprising a variable region,also known as, a sFv; a disulfide-linked Fv, or dsFv; a camelized VH; abispecific sFv; a diabody; and a triabody. Derivatives of antibodiesalso include one or more CDR sequences of an antibody combining site. Incertain embodiments, the antibody to be used with the inventioncomprises a single-chain Fv (“scFv”).

The antibodies used in the methods of the invention may be from anyanimal origin including birds and mammals (e.g., human, murine, donkey,sheep, rabbit, goat, guinea pig, camel, horse, or chicken).

In certain embodiments, the antibodies of the invention are monoclonalantibodies (mAbs). Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technologies, or a combination thereof.For example, mAbs can be produced using hybridoma techniques includingthose known in the art and taught, for example, in Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563 681 (Elsevier, N.Y., 1981) (each of which is hereinincorporated by reference in their entireties).

Antibodies can also be generated using various phage display methods.Examples of phage display methods that can be used to make theantibodies include those disclosed in Brinkman et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology57:191-280; PCT Application No. PCT/GB91/O1 134; InternationalPublication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,5,733,743 and 5,969,108; each of which is incorporated by referenceherein in its entirety.

In certain embodiments, the antibodies of the invention are chimericantibodies or single chain antibodies. Techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, Proc NatlAcad Sci 81:851; Neuberger et al., 1984 Nature 312:604; Takeda et al.,1985, Nature 314:452, each incorporated by reference herein in itsentirety) and single chain antibodies (U.S. Pat. No. 4,946,778; Bird,1988, Science 242:423; Huston et al, 1988, Proc Natl Acad Sci USA85:5879; and Ward et al, 1989, Nature 334:544, each incorporated byreference herein in its entirety) are well known in the art.

In a certain embodiment, antibodies used in the methods of the inventionare humanized antibodies. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International publicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089,each of which is herein incorporated by reference in its entirety),veneering or resurfacing (European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering 7(6):805-814; and Roguska et al, 1994,PNAS 91:969-973, each of which is incorporated by reference herein inits entirety), chain shuffling (U.S. Pat. No. 5,565,332, hereinincorporated by reference in its entirety), and techniques disclosed in,e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, WO 9317105, Tan et al., 2002,J. Immunol. 169:1119 25, Caldas et al., 2000 Protein Eng. 13(5):353-60,Morea et al., 2000, Methods 20(3):267 79, Baca et al., 1997, J. Biol.Chem. 272(16):10678-84, Roguska et al., 1996, Protein Eng. 9(10):895904, Couto et al, 1995 Cancer Res. 55 (23 Supp):5973s-5977s, Couto etal., 1995, Cancer Res. 55(8):1717-22, Sandhu J S, 1994, Gene150(2):409-10, and Pedersen et al., 1994, J. Mol. Biol. 235(3):959-73U.S. Patent Pub. No. US 2005/0042664 A1 (Feb. 24, 2005), each of whichis incorporated by reference herein in its entirety. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Reichmann et al., 1988, Nature332:323, each of which is incorporated by reference herein in itsentirety).

Single domain antibodies can be produced by methods well-known in theart. (See, e.g., Riechmann et al., 1999, J. Immunol. 231:25-38; Nuttallet al., 2000, Curr. Pharm. Biotechnol. 1(3):253-263; Muylderman, 2001,J. Biotechnol. 74(4):277302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591, and WO 01/44301, each ofwhich is incorporated herein by reference in its entirety).

Further, antibodies that bind to a desired antigen can, in turn, beutilized to generate anti-idiotype antibodies that “mimic” an antigenusing techniques well known to those skilled in the art. (See, e.g.,Greenspan & Bona, 1989, FASEB J. 7(5):437-444; and Nissinoff, 1991, J.Immunol. 147(8):2429-2438, herein incorporated by reference in theirentireties).

Bispecific antibodies can be prepared using techniques that are known inthe art. (See, e.g., U.S. Pat. Nos. 5,534,254, 5,837,242, 6,492,123;U.S. patent application publication Nos. 20040058400 and 20030162709,which are all herein incorporated by reference in their entireties).

The present invention contemplates the use of antibodies recombinantlyfused or chemically conjugated (including both covalently andnon-covalently conjugations) to a polypeptide. Fused or conjugatedantibodies of the present invention may be used for ease inpurification. For example, the antibodies or fragments thereof for usein the present invention can be fused to marker sequences, such as apeptide to facilitate purification. See e.g., PCT publication WO93/21232; EP 439,095; Naramura et al., 1994, Immunol Lett 39:91; U.S.Pat. No. 5,474,981; Gillies et al., 1992, Proc Natl Acad Sci USA89:1428; Fell et al., 1991, J Immunol 146:2446, which are hereinincorporated by reference in their entireties.

In certain aspects, the antibodies used in the present invention can beproduced intracellularly, in the periplasmic space, or directly secretedinto the medium. If the antibodies are produced intracellularly, as afirst step, the particulate debris, either host cells or lysedfragments, may be removed, for example, by centrifugation orultrafiltration.

Exemplary methods for the use of host cells and vectors in theproduction of antibody can be found in U.S. Pat. Nos. 4,816,567 and6,331,415 to Cabilly et al., each of which is incorporated by referenceherein in its entirety.

5.5 Therapeutic Applications of the B10 Cell Subset to Treat Diseasesand Disorders Associated with Diminished IL-10 Levels

Diseases or disorders associated with diminished levels of IL-10 andelevated immune/inflammatory responses (particularly inflammatorydiseases and autoimmune diseases) can be treated in accordance with theinvention using different therapeutic modalities designed to supply theB10 cell subset to an affected subject (e.g., by adoptivetransfer/transplant); expand the endogenous B10 cell subset in anaffected subject; and/or enhance production of IL-10 by the B10 cellsubset (either adoptively transferred cells or the endogenouspopulation) in the affected subject.

In one approach, a cellular composition enriched for the B10 cell subsetis administered to a subject in need thereof in amounts effective toincrease IL-10. The cellular composition can be obtained from ahistocompatibilty matched donor. Alternatively, lymphocytes may beobtained from the subject to be treated, enriched for the B10 cellsubset and returned to the patient. In either case the enriched cellscan be exposed to an antigen of interest prior to introduction into thesubject to further fine-tune the regulation of the immune response. Theenriched or selected B10 cells may also be expanded by stimulation invitro prior to introduction into the subject.

Alternatively, an effective amount of a therapeutic agent capable ofstimulating the proliferation of the endogenous B10 cell subset, and/orincreasing the amounts of IL-10 produced by the B10 cell subset can beadministered to a subject in need thereof in amounts effective toincrease IL-10 levels in said subject. These agents may be targeted tothe B10 cell cell subset. Such agents include CD40 agonists and TLRagonists.

5.5.1. Diseases and Disorders Associated with Reduced IL-10 Productionthat can be Treated Using the B10 Cell Subset

Diseases and conditions associated with diminished IL-10 levels can betreated in accordance with this aspect of the invention. Decreasedlevels of IL-10 have been demonstrated in autoimmune and inflammatorydiseases including, but not limited to psoriasis (Asadullah et al.,1998, J. Clin. Investig. 101:783-94, Nickoloff et al., 1994, Clin.Immunol. Immunopathol., 73:63-8, Mussi et al. 1994, J. Biol. Regul.Homeostatic Agents), rheumatoid arthritis (Jenkins et al., 1994,Lymphokine Cytokine Res. 13:47-54; Cush et al., 1995, Arthritis Rheum.38:96-104; Al Janadi et al., 1996, J. Clin. Immunol. 16:198-207),allergic contact dermatitis (Kondo et al., 1994, J. Investig. Dermatol.103:811-14; Schwarz et al., 1994, J. Investig. Dermatol. 103:211-16),inflammatory bowel disease (Kuhn et al., 1993, Cell 75:263-74; Lindsayand Hodgson, 2001, Aliment. Pharmacol. Ther. 15:1709-16) and multiplesclerosis (Barrat et al., 2002, J. Exp. Med. 195:603-16; Cua et al.,2001, J. Immunol. 166:602-8; Massey et al., 2002, Vet. Immunol.Immunopathol. 87:357-72; Link and Xiao, 2001, Immunol. Rev. 184:117-28).

Any type of autoimmune disease can be treated in accordance with thismethod of the invention. Non-limiting examples of autoimmune disordersinclude: alopecia areata, ankylosing spondylitis, antiphospholipidsyndrome, autoimmune Addison's disease, autoimmune diseases of theadrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune oophoritis and orchitis, autoimmune thrombocytopenia,Behcet's disease, bullous pemphigoid, cardiomyopathy, celiacsprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy, Churg-Strausssyndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinindisease, Crohn's disease, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis,Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathicpulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgAneuropathy, juvenile arthritis, lichen planus, lupus erthematosus,Méniere{grave over (')}s disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteritis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein, someautoimmune disorders are associated with an inflammatory condition.Thus, there is overlap between what is considered an autoimmune disorderand an inflammatory disorder. Therefore, some autoimmune disorders mayalso be characterized as inflammatory disorders.

In an aspect of this embodiment, the methods of the invention can beused to treat inflammatory diseases associated with diminished IL-10levels, but not autoimmune diseases.

In another aspect of this embodiment, the methods of the invention canbe used to treat autoimmune diseases associated with diminished IL-10levels, but not inflammatory diseases.

In yet another aspect of this embodiment, the methods of the inventioncan be used to treat autoimmune diseases associated with diminishedIL-10 levels, wherein the autoimmune disease to be treated is notsystemic lupus erythematosus.

Any type of inflammatory disease can be treated in accordance with thismethod of the invention. Non-limiting examples of inflammatory diseasesinclude, but are not limited to, asthma, encephilitis, inflammatorybowel disease, chronic obstructive pulmonary disease (COPD), allergicdisorders, septic shock, pulmonary fibrosis, undifferentiatedspondyloarthropathy, undifferentiated arthropathy, arthritis,inflammatory osteolysis, and chronic inflammation resulting from chronicviral or bacteria infections.

In still another aspect of this embodiment, the methods of the inventionencompass therapies that are aimed at treating diseases associated witha helper T (Th) 1-mediated inflammatory response but not diseasesassociated with a Th2-mediated inflammatory response.

In an alternative aspect of this embodiment, the methods of theinvention encompass therapies that are aimed at treating diseasesassociated with a Th2-mediated inflammatory response but not diseasesassociated with a Th1-mediated inflammatory response.

IL-10 is capable of inhibiting ischemia/reperfusion injury (Deng et al.,2001, Kidney Int. 60:2118-28), graft-versus-disease, andtransplant-related mortality (Baker et al., 1999, Bone Marrow Transplant23:1123-9; Holler et al., 2000, Bone Marrow Transplant 25:237-41). Assuch, one embodiment of the present invention involves treatingtransplant-associated diseases/conditions by increasing the level ofIL-10 in a patient in need thereof.

In another embodiment, the levels of endogenous IL-10 are increased in asubject receiving an organ transplant by administration of a B10 cellsubset. In one aspect of this embodiment, the B10 cell population isisolated from the patient themselves, i.e., the subject is the donor. Inanother aspect of this embodiment, the B10 cell population is isolatedfrom a donor that is not the subject. The donor of the B10 cells may bethe same as the organ donor. In another embodiment, the B10 cellpopulation is pooled from several donors.

5.5.2. Therapeutic Modalities

In one embodiment, a subject suffering from an autoimmune disease or aninflammatory disease associated with diminished levels of IL-10 isadministered a population of B10 cells. In one aspect of thisembodiment, the B10 cell population is isolated from the patientthemselves, i.e., the subject is the donor. In another aspect of thisembodiment, the B1U cell population is isolated from a donor that is notthe subject. In an aspect of this embodiment, the B10 cell population ispooled from several donors. According to this embodiment, administrationof a B10 cell population to a subject in need thereof results in anincreased level of IL-10 production in the patient sufficient tocontrol, reduce, or eliminate symptoms of the disease being treated.

In one aspect of this embodiment, the therapeutic agent is an antibody,in particular, a CD40 antibody or a CD40 agonist. In other aspects, thetherapeutic agent is a small molecule, a polypeptide, DNA, or RNA thatinteracts with the B cell CD40 receptor. In another embodiment thetherapeutic agent is a TLR agonist, such as LPS or CpG. The therapeuticagent may be a different TLR agonist or a small molecule, polypeptide,RNA, dsRNA, or DNA capable of interacting with a TLR receptor.

In another embodiment, a subject suffering from an inflammatory orautoimmune disease associated with diminished levels of IL-10 is treatedby administration of a therapeutic agent capable of causing an increasein IL-10 production by the B10 cells in the patient. In a specificaspect of this embodiment, the therapeutic agent targets the B cell CD40receptor or a TLR. In another aspect of this embodiment, the therapeuticagent is a CD40 antibody a TLR agonist, a small molecule, a polypeptide,DNA, or RNA that is capable of binding, targeting, and or modulatingCD40 so as to result in an increase in IL-10 production by the B10 cellsin the subject.

An antibody according to these embodiments can be any type of antibodyor fragment thereof, as described above. According to this embodimentadministration of a CD40 antibody or fragment thereof to a subject withan autoimmune disease or an inflammatory disease associated withdiminished levels of IL-10 results in an upregulation of IL-10production by the endogenous B10 cell population in the subject.

In still another embodiment, a patient receiving a transplant isadministered a therapeutic agent capable of increasing endogenous IL-10production by the B10 cell subset of that patient to increase thepatient's tolerance to the transplant. In yet another embodiment, apatient receiving a transplant is administered a B10 cell subset toincrease the patient's tolerance to the transplant.

The subject is preferably a mammal such as non-primate (e.g., cows,pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey, suchas a cynomolgous monkey and a human). In a preferred embodiment, thesubject is a human.

5.5.2.1. B10 Cells as Therapeutic Agents

In one embodiment, adoptive transfer of B10 cells can be effective tosuppress a wide variety of diseases, including, but not limited to anyof those described above, I.e., autoimmune diseases, inflammatorydiseases, or any other disease which may be treated by introduction of aB10 cell population into a subject. Adoptive transfer of B10 cells canfurther be employed to minimize the immune/inflammatory responseassociated with transplant of cells and/or tissues.

In an exemplary adoptive cell transfer protocol, a mixed population ofB10 cells is initially extracted from a target donor. The B10 cellsisolated from the donor may be isolated from any location in the donorin which they reside including, but not limited to, the blood, spleen,lymph nodes, and/or bone marrow of the donor. Depending on theapplication, the B 10 cells may be extracted from a healthy donor, adonor suffering from a disease that is in a period of remission orduring active disease; or from the organs, blood, or tissues of a donorthat has died. In the case of the latter, the donor is an organ donor.In yet another embodiment, the B10 cells can be obtained from thesubject, expanded or activated and returned to the subject.

Harvested lymphocytes may be separated by flow cytometry or other cellseparation techniques based on B10 cell markers such as those describedherein (e.g., CD1d, CD5, CD24, and CD27), and then transfused to arecipient. Alternatively, the cells may be stored for future use. In oneaspect of this embodiment, the donor and the recipient are the samesubject. In another aspect of this embodiment, the donor is a subjectother than the recipient. In a further aspect of this embodiment, the“donor” comprises multiple donors other than the recipient, wherein theB10 cells from said multiple donors are pooled.

In another embodiment, the B10 cell population obtained from a donor canbe expanded, enriched, or made to produce elevated levels of IL-10, asdescribed in sections 5.1 and 5.2, supra, prior to being administered toa recipient.

In the adoptive transfer techniques contemplated herein, wherein thedonor is a subject other than the recipient, the recipient and the donorare histocompatible. Histocompatibility is the property of having thesame, or mostly the same, alleles of a set of genes called the majorhistocompatibility complex (MHC). These genes are expressed in mosttissues as antigens, to which the immune system makes antibodies. Whentransplanted cells and/or tissues are rejected by a recipient, the bulkof the immune system response is to the MHC proteins. MHC proteins areinvolved in the presentation of foreign antigens to T-cells, andreceptors on the surface of the T-cell are uniquely suited torecognition of proteins of this type. MHC are highly variable betweenindividuals, and therefore the T-cells from the host recognize theforeign MHC with a very high frequency leading to powerful immuneresponses that cause rejection of transplanted tissue. As such, thechance of rejection of the B10 cell population by the recipient isminimized.

The amount of B10 cells which will be effective in the treatment and/orsuppression of a disease or disorder which may be treated byintroduction of a B10 cell population into a subject can be determinedby standard clinical techniques. The dosage will depend on the type ofdisease to be treated, the severity and course of the disease, thepurpose of introducing the B10 cell population, previous therapy therecipient has undertaken, the recipient's clinical history, and thediscretion of the attending physician. The B10 cell population can beadministered in treatment regimes consistent with the disease, e.g., asingle or a few doses over one to several days to ameliorate a diseasestate or periodic doses over an extended time to inhibit diseaseprogression and prevent disease recurrence. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses may be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Exemplary, non-limiting doses that could be used in the treatment ofhuman subjects range from at least 3.8×10⁴, at least 3.8×10⁵, at least3.8×10⁶, at least 3.8×10⁷, at least 3.8×10⁸, at least 3.8×10⁹, or atleast 3.8×10¹⁰B10 cells/m². Ina certain embodiment, the dose used in thetreatment of human subjects ranges from about 3.8×10⁹ to about 3.8×10¹⁰B10 cells/m².

In another aspect of this embodiment, the B10 cells obtained from thedonor can be introduced into a recipient at a desired location, so as tospecifically target the therapeutic effects of the B10 cell population,i.e., IL-10 production. Such techniques can be accomplished usingimplantable immune modulation devices, e.g., virtual lymph nodes, suchas those described in U.S. patent application publication No.2003/0118630; WO1999/044583; and U.S. Pat. No. 6,645,500, which areincorporated by reference herein in their entireties. According to thisembodiment, a B10 cell population can be isolated from a donor asdescribed above, added to an implantable immune modulation device, andsaid device then can be inplanted into a recipient at a location wherethe therapeutic effects of the B10 cell population, i.e., IL-10production, are needed.

5.5.2.2. Antigen-Specific B10 Cells

In another embodiment, the B10 cell population can be made responsive toa certain antigen involved in a specific disease. In an aspect of thisembodiment, the B10 cell population, when sensitized with a certainantigen, will produce therapeutic amounts of IL-10 upon subsequentencounters with the antigen. In an aspect of this embodiment, such anantigen-specific B10 cell population may be used in an adoptive transfertechnique, wherein a subject is or has previously been immunized with acertain antigen and the antigen-sensitized B10 cells from said subjectare isolated and transplanted to the same or another subject. In stillanother aspect of this embodiment, a B10 cell population from a subjectcan be isolated and subsequently can be sensitized with adisease-specific antigen ex vivo or in vitro. The sensitized B10 cellpopulation can then be transplanted into the original or another subjectby any method known in the art. In still another aspect of thisembodiment, the antigen-specific B10 cell population can be added to animplantable immune modulation device, as described above. According tothis embodiment, the implanted B10 cell population will producestrategically localized IL-10 when encountering antigen in the host. Ina further aspect, the B10 cell population and a disease-specific antigencan both be placed in an implantable immune modulation device, and saiddevice then can be transplanted into a recipient at a location where thetherapeutic effects of the B10 cell population, i.e., IL-10 production,are needed, thus resulting in an amplified response to the disease invivo.

In another aspect, a certain disease-specific antigen can beadministered in conjunction with a CD40 agonist or a TLR agonist. In acertain aspect of this embodiment, the therapeutic agent is an antibody,in particular, a CD40 antibody or LPS or CpG oligodeoxynucleotides. Inother aspects, the therapeutic agent is a small molecule, a polypeptide,DNA, or RNA that interacts with the B cell CD40 receptor or TLRs.

Any antigen from any disease, disorder, or condition may be used inaccordance with the methods of the invention. Exemplary antigens includebut are not limited to bacterial, viral, parasitic, allergens,autoantigens and tumor-associated antigens. If a DNA based vaccine isused the antigen will typically be encoded by a sequence of theadministered DNA construct. Alternatively, if the antigen isadministered as a conjugate the antigen will typically be a proteincomprised in the administered conjugate. Particularly, the antigen caninclude protein antigens, peptides, whole inactivated organisms, and thelike.

Specific examples of antigens that can be used in the invention includeantigens from hepatitis A, B, C or D, influenza virus, Listeria,Clostridium botulinum, tuberculosis, tularemia, Variola major(smallpox), viral hemorrhagic fevers, Yersinia pestis (plague), HIV,herpes, pappilloma virus, and other antigens associated with infectiousagents. Other antigens include antigens associated with autoimmuneconditions, inflammatory conditions, allergy, and asthma. Non-limitingexamples of autoimmune diseases and inflammatory diseases are provided,supra.

In an aspect of this embodiment, a B10 cell population sensitized with adisease-specific antigen can be administered alone or in conjunctionwith a CD40 agonist or TLR, in particular, a CD40 antibody, for use as atherapeutic or prophylactic vaccine for conferring immunity against suchdisease conditions.

In another embodiment, a B10 cell subset may be sensitized with antigenfrom a prospective transplant donor, so as to increase the levels ofIL-10 production by the B10 cells in a transplant recipient. In anaspect of this embodiment, the increased IL-10 production by the B10cell subset in the transplant recipient results in a decreasedimmune/inflammatory response to the transplant in the transplantrecipient. The role of B10 cells in transplants is described in section5.5.2.3, infra.

5.5.23. B10 Cells in Organ Transplant Patients

In another embodiment, the levels of endogenous IL-10 are increased in asubject receiving an organ transplant by administration of a B10 cellsubset. In one aspect of this embodiment, the B10 cell population isisolated from the patient themselves, i.e., the subject is the donor. Inanother aspect of this embodiment, the B10 cell population is isolatedfrom a donor that is not the subject. In an aspect of this embodiment,the B10 cell population is pooled from several donors. In another aspectof this embodiment, the B10 cell subset is isolated from a subject thathas died, wherein said subject is an organ donor. In embodiments whereinthe B10 cells are from a donor that is not the subject, the subject andthe donor are histocompatible.

In one aspect of this embodiment, the B10 cell subset is administeredprior to the transplant. According to this aspect, the therapeutic agentcan be administered at least 1 hour, at least 12 hours, at least 1 day,at least 2 days, at least 3 days, at least 4 days, at least 5 days, atleast 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, or at least 1 month prior to the transplant.Administration of the therapeutic agent can be by any method known tothose of skill in the art.

In another aspect of this embodiment, the B10 cell subset isadministered at the same time as the transplant.

In still another aspect of this embodiment, the B10 cell subset isadministered following the transplant.

In a certain aspect, the B10 cell subset is administered before, during,and after the transplant. According to this aspect, when the B10 cellsubset is administered after the transplant, it may be administered forat least 12 hours, at least 1 day, at least 2 days, at least 3 days, atleast 4 days, at least 5 days, at least 6 days, at least 1 week, atleast 2 weeks, at least 3 weeks, at least 4 weeks, least 1 month, or atleast 1 year following the transplant or for the duration of thepatient's life.

As described in section 5.5.2.2, supra, in one embodiment, a B10 cellsubset administered to a patient that is receiving a transplant can besensitized with antigens specific to the transplanted material.According to this embodiment, the transplant recipient will have adecreased immune/inflammatory response to the transplanted material and,as such, the likelihood of rejection of the transplanted material isminimized.

In another embodiment, the levels of endogenous IL-10 are increased in asubject receiving an organ transplant by administration of a therapeuticagent capable of causing an increase in IL-10 production by the B10cells in the patient. The therapeutic agent can be administered in vivoor ex vivo; i.e., the B10 cell population can be isolated/enriched fromthe patient, contacted with the therapeutic agent ex vivo, and the“activated” population returned to the patient. In a specific aspect ofthis embodiment, the therapeutic agent targets the B cell CD40 receptoror a TLR. In another aspect of this embodiment, the therapeutic agent isa CD40 antibody, a small molecule, a polypeptide, DNA, or RNA that iscapable of binding, targeting, and or modulating CD40 so as to result inincrease in IL-10 production by the B10 cells in the subject.

In one aspect of this embodiment, the therapeutic agent capable ofcausing an increase in IL-10 production by the B10 cells in the patientis administered prior to the transplant. According to this aspect, thetherapeutic agent can be administered at least 1 hour, at least 12hours, at least 1 day, at least 2 days, at least 3 days, at least 4days, at least 5 days, at least 6 days, at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, or at least 1 month prior tothe transplant. Administration of the therapeutic agent can be by anymethod known to those of skill in the art.

In another aspect of this embodiment, the therapeutic agent capable ofcausing an increase in IL-10 production by the B10 cells in the patientis administered at the same time as the transplant.

In still another aspect of this embodiment, the therapeutic agentcapable of causing an increase in IL-10 production by the B10 cells inthe patient is administered following the transplant.

In a certain aspect, the therapeutic agent capable of causing anincrease in IL-10 production by the B10 cells in the patient isadministered before, during, and after the transplant. According to thisaspect, when the therapeutic agent capable of causing an increase inIL-10 production by the B10 cells in the patient is administered afterthe transplant, it may be administered for at least 12 hours, at least 1day, at least 2 days, at least 3 days, at least 4 days, at least 5 days,at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, atleast 4 weeks, least 1 month, or at least 1 year following thetransplant or for the duration of the patient's life.

According to these embodiments, administration of a therapeutic agentcapable of causing an increase in IL-10 production by the B10 cells inthe patient or administration of a B10 cell subset results in adecreased immune response in the patient receiving the transplant,wherein the decreased immune response results in an increased likelihoodthat the transplant will be accepted, an increased tolerance to thetransplant, an increased duration of time in which the transplant isaccepted, and/or an increased lifespan in the transplant recipient.

Any type of transplant can be performed according to these methods.

5.6 Therapeutic Targeting of the B Cell Subset to Treat Diseases andDisorders Associated with Enhanced IL-10 Levels

In another embodiment, the invention provides methods for treatingand/or managing a disease or disorder associated with adecreased/depressed/impaired immune/inflammatory response, particularlycancer or an infectious disease, by administrating to a subject in needthereof a therapeutically or prophylactically effective amount of atherapeutic agent capable of ablating the population of B10 cells and/orthe amounts of IL-10 being produced by the B10 cell subset. In anotherembodiment, the invention provides methods for the treatment of cancerby administrating to a subject in need thereof a therapeutically orprophylactically effective amount of a therapeutic agent capable ofablating the population of B10 cells and/or the amounts of IL-10 beingproduced by the B10 cell subset.

In an aspect of this embodiment, the therapeutic agent is an antibodythat mediates CDC or ADCC and kills target cells, or an immunoconjugatethat alters the function of or kills target cells is used. Inparticular, a CD22 mAb that kills or inhibits the proliferation of theB10 cell subset can be used. Alternatively, a CD1d, a CD5, a CD24, or aCD27 antibody or a bispecific a CD1d/CD5 or a CD24/CD27 antibody can beused.

In another aspect of this embodiment, the therapeutic agent is anantibody that does not utilize CDC or ADCC to kill the target cells. Inanother aspect, the antibody does not kill the target cells byapoptosis.

In another aspect of this embodiment, the therapeutic agent is anantibody that does not utilize CDC, ADCC, or apoptosis as the primarymechanism for killing target cells, I.e., the majority of target cellsare killed by a mechanism that is CDC-, ADCC-, andapoptosis-independent.

In another aspect of this embodiment, the therapeutic agent is a smallmolecule, a polypeptide, DNA, or RNA that interacts with the B cell CD22receptor or with CD1d, CD5, CD20, CD24, or CD27.

The subject is preferably a mammal such as non-primate (e.g., cows,pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkey, suchas a cynomolgous monkey and a human). In a preferred embodiment, thesubject is a human.

5.6.1 Diseases and Disorders Associated with Increased IL-10 Production

IL-10 has been shown to promote tumor growth and overexpression of IL-10has been demonstrated in certain cancers (Matsuda et al., 1994, J. Exp.Med. 180:2371-6; Salazar-Onfray et al., 1997, J. Immunol. 159:3195-3202;Hagenbaugh et al. 1997, J. Exp. Med. 185:2101-110; Kruger-Kraskagakes etal. 1994, Br. J. Cancer 70:1182-5, Dummer et al., 1996, Int. J. Cancer66:607-10; Kim et al., 1995, J. Immunol. 155:2240-47; Blay et al., 1993,Blood 82:2169-74; Asadullah et al., 2000, Exp. Dermatol. 9:71-6). Assuch, one embodiment of the present invention involves treating cancerby decreasing the level of IL-10 in a patient in need thereof byablation of the B10 cell subset and/or reducing the amount of IL-10produced by the B10 cell subset.

Any type of cancer can be treated in accordance with this method of theinvention. Non-limiting examples of cancers include: leukemias, such asbut not limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias, such as, myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia leukemias andmyelodysplastic syndrome; chronic leukemias, such as but not limited to,chronic myelocytic (granulocytic) leukemia, chronic lymphocyticleukemia, hairy cell leukemia; polycythemia vera; lymphomas such as butnot limited to Hodgkin's disease, non-Hodgkin's disease; multiplemyelomas such as but not limited to smoldering multiple myeloma,nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia,solitary plasmacytoma and extramedullary plasmacytoma; Waldenström'smacroglobulinemia; monoclonal gammopathy of undetermined significance;benign monoclonal gammopathy; heavy chain disease; bone and connectivetissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor,fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limitedto, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, primary brain lymphoma; breast cancer including but notlimited to ductal carcinoma, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer, adrenal cancer such as but notlimited to pheochromocytom and adrenocortical carcinoma; thyroid cancersuch as but not limited to papillary or follicular thyroid cancer,medullary thyroid cancer and anaplastic thyroid cancer; pancreaticcancer such as but not limited to, insulinoma, gastrinoma, glucagonoma,vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor,pituitary cancers such as but limited to Cushing's disease,prolactin-secreting tumor, acromegaly, and diabetes insipius; eyecancers such as but not limited to ocular melanoma such as irismelanoma, choroidal melanoma, and cilliary body melanoma, andretinoblastoma; vaginal cancers such as squamous cell carcinoma,adenocarcinoma, and melanoma; vulvar cancer such as squamous cellcarcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, andPaget's disease; cervical cancers such as but not limited to, squamouscell carcinoma, and adenocarcinoma; uterine cancers such as but notlimited to endometrial carcinoma and uterine sarcoma; ovarian cancerssuch as but not limited to, ovarian epithelial carcinoma, borderlinetumor, germ cell tumor, and stromal tumor, esophageal cancers such asbut not limited to, squamous cancer, adenocarcinoma, adenoid cysticcarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell)carcinoma; stomach cancers such as but not limited to, adenocarcinoma,fungating (polypoid), ulcerating, superficial spreading, diffuselyspreading, malignant lymphoma, liposarcoma, fibrosarcoma, andcarcinosarcoma; colon cancers; rectal cancers; liver cancers such as butnot limited to hepatocellular carcinoma and hepatoblastoma; gallbladdercancers such as adenocarcinoma; cholangiocarcinomas such as but notlimited to papillary, nodular, and diffuse; lung cancers such asnon-small cell lung cancer, squamous cell carcinoma (epidermoidcarcinoma), adenocarcinoma, large-cell carcinoma and small-cell lungcancer, testicular cancers such as but not limited to germinal tumor,seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma,embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sactumor), prostate cancers such as but not limited to, prostaticintraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers such as but notlimited to renal cell carcinoma, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor, bladder cancers such as but not limited to transitionalcell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Inaddition, cancers include myxosarcoma, osteogenic sarcoma,endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma,hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogeniccarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillarycarcinoma and papillary adenocarcinomas (for a review of such disorders,see Fishman et al, 1985, Medicine, 2d Ed., J. B. Lippincott Co.,Philadelphia and Murphy, 1997, Informed Decisions: The Complete Book ofCancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin BooksU.S.A., Inc., United States of America, incorporated by reference hereinin its entirety).

Increased levels of IL-10 have been demonstrated in certain autoimmuneand inflammatory diseases including, but not limited to systemic lupuserythematosus (Park et al., 1998, Clin. Exp. Rheumatol. 16:283-88;Llorente et al., 1995, J. Exp. Med. 181:839-44), systemic sclerosis(Hasegawa et al., 1997, J. Rheumatol. 24:328-32), Bullous Pemphigoid(Schmidt et al., 1996, Arch. Dermatol. Res. 228:353-7; Giacalone et al.,1998, Exp. Dermatol. 7:157-61), and atopic dermatitis (Ohmen et al.,1995, J. Immunol. 154:1956-63; Asadullah et al., 1996, J. Investig.Dermatol. 197:833-7). As such, one embodiment of the present inventioninvolves treating an autoimmune or inflammatory by decreasing the levelof IL-10 in a patient in need thereof by ablation of the B10 cell subsetand/or reducing the amount of IL-10 produced by the B10 cell subset.

Any type of autoimmune disease that is accompanied by increased IL-10production can be treated in accordance with this method of theinvention. A non-limiting list of autoimmune disorders is providedabove.

Any type of inflammatory disease that is accompanied by increased IL-10production can be treated in accordance with this method of theinvention. A non-limiting list of inflammatory diseases is providedabove.

In an aspect of this embodiment, the methods of the invention can beused to treat inflammatory diseases associated with diminished IL-10levels, but not autoimmune diseases.

In another aspect of this embodiment, the methods of the invention canbe used to treat autoimmune diseases associated with diminished IL-10levels, but not inflammatory diseases.

In yet another aspect of this embodiment, the methods of the inventioncan be used to treat autoimmune diseases associated with diminishedIL-10 levels, wherein the autoimmune disease to be treated is notsystemic lupus erythematosus.

In still another aspect of this embodiment, the methods of the inventionencompass therapies that are aimed at treating diseases associated witha helper T (Th) 1-mediated inflammatory response but not diseasesassociated with a Th2-mediated inflammatory response.

In an alternative aspect of this embodiment, the methods of theinvention encompass therapies that are aimed at treating diseasesassociated with a Th2-mediated inflammatory response but not diseasesassociated with a Th1-mediated inflammatory response.

IL-10 has also been shown to be associated with the pathogenesis orineffective immune response to many infectious diseases as well. Theinfectious diseases may be mediated by viruses, bacteria, yeast,parasites or prions. A non-limiting list of infectious diseases that maybenefit from decreasing or inhibiting IL-10 production by the B10 cellsubset includes, but is not limited to Diphtheria, Tetanus, Pertussis,Haemophilus influenzae type b, Anthrax, Measles, Rubella, Mumps,Botulism, Chickenpox, Cholera, Hepatitis B, Influenza, Hepatitis A,Hepatitis C, Rabies, Polio, Japanese Encephalitis Virus, Meningitis,Typhoid, Pneumonia, Rocky Mountain Spotted Fever, Lyme Disease,Smallpox, Tetanus, Mycobacterium, Malaria, HIV/AIDS, RSV, Herpesviruses,and Yellow Fever.

5.6.2 Therapies

In one embodiment, a subject suffering from cancer who has elevatedlevels of IL-10 is treated by administration of a therapeutic agentcapable of ablating the population of B10 cells in the patient and/orreducing the amount of IL-10 produced by the B10 cell population. In aspecific aspect of this embodiment, the therapeutic agent targets the Bcell CD22 receptor. In another aspect of this embodiment, thetherapeutic agent is a CD22 antibody, a small molecule, a polypeptide,DNA, or RNA that is capable of binding, targeting, and or modulatingCD22 so as to result in ablation of the B10 cell subset. In a specificaspect of this embodiment, the therapeutic agent targets the B cell CD20receptor. In another aspect of this embodiment, the therapeutic agent isa CD20 antibody, a small molecule, a polypeptide, DNA, or RNA that iscapable of binding, targeting, and or modulating CD20 so as to result inablation of the B10 cell subset.

In another embodiment, a subject suffering from an immune deficiencydisease associated with elevated levels of IL-10 is treated byadministration of a therapeutic agent capable of ablating the populationof B10 cells in the patient and thereby reducing the amount of IL-10produced by the B10 cell population. Ina specific aspect of thisembodiment, the therapeutic agent targets the B cell CD22 receptor. Inanother aspect of this embodiment, the therapeutic agent is a CD22antibody, a small molecule, a polypeptide, DNA, or RNA that is capableof binding, targeting, and or modulating CD22 so as to result inablation of the B10 cell subset.

In an alternative embodiment, a subject suffering from cancer or animmune deficiency disease associated with elevated levels of IL-10 istreated by administration of a CD1d, CD5, CD24, CD27 antibody or abispecific CD1d/CD5 or CD24/CD27 antibody capable of ablating thepopulation of B10 cells in the patient and thereby reducing the amountof IL-10 produced.

In order to kill or ablate the B10 cell subset, targeting antibodies ofan isotype that mediate ADCC (antibody-dependent and mediated toxicity)or CDC (complement-dependent cytotoxicity) can be used. Of the varioushuman immunoglobulin classes, IgG1, IgG2, IgG3, IgG4 and IgM are knownto activate complement. Human IgG1 and IgG3 are known to mediate ADCC.Antibodies to CD20 may be used to deplete B10 cells selectively. Theantibodies demonstrated to target the IL-10 producing B10 cell subsetwere antibodies that were not capable of inducing ADCC, CDC orapoptosis. Instead the effective antibodies were those that inducedhomotypic adhesion. Thus, antibodies to B cell surface markers with IgG3or IgG2b Fc regions which do not efficiently engage most Fey receptors,but induce homotypic adhesion are useful in the methods describedherein. Optionally the Fc portion of a known antibody may be modified sothat the mechanism of depletion of B10 cells is independent of theantibody's Fc region.

Antibodies targeting the B10 cell subset can be further conjugated to acytotoxic agent, using methods known in the art (see, e.g., DiJoseph etal., 2004, Clin. Cancer Res. 10:8620-9). This may be preferred whenusing antibodies or antibody fragments that do not mediate ADCC or CDC.Non-limiting examples of cytotoxic agents include antimetabolites (e.g.,cytosine arabinoside, aminopterin, methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, and 5-fluorouracil decarbazine); alkylatingagents (e.g., mechlorethamine, thiotepa chlorambucil, melphalan,carmustine (BCNU) and lomustine (CCNU), cyclophosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiammine-platinum (II) (CDDP), and cisplatin); vincaalkaloid; anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); calicheamicin; CC-1065and derivatives thereof; auristatin molecules (e.g., auristatin PHE,bryostatin-1, and dolastatin-10; see Woyke et al., Antimicrob. AgentsChemother 46:3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother.45:3580-4 (2001), Mohammad et al., Anticancer Drugs 12:735-40 (2001),Wall et al., Biochem. Biophys. Res. Commun. 266:76-80 (1999), Mohammad,et al., Int. J. Oncol. 15:367-72 (1999), all of which are incorporatedby reference herein in their entireties); DNA-repair enzyme inhibitors(e.g., etoposide or topotecan); kinase inhibitors (e.g., compoundST1571, imatinib mesylate (Kantarjian et al., Clin. Cancer Res.8(7):2167-76 (2002)); demecolcine; and other cytotoxic agents (e.g.,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracenedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof and those compounds disclosed in U.S. Pat. Nos.6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459, all of which are incorporated byreference herein in their entirety); farnesyl transferase inhibitors(e.g., R115777, BMS-214662, and those disclosed by, for example, U.S.Pat. Nos. 6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959,6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615,6,387,905, 6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487,6,300,501, 6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338,6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786,6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465,6,124,295, 6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853,6,071,935, 6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and6,040,305, all of which are herein incorporated by reference in theirentirety); topoisomerase inhibitors (e.g., camptothecin, irinotecan,SN-38, topotecan, 9-aminocamptothecin, GG211 (GI147211), DX-8951f,IST-622, rubitecan, pyrazoloacridine, XR5000, saintopin, UCE6, UCE1022,TAN-1518A, TAN 1518B, KT6006, KT6528, ED-110, NB-506, ED-110, NB-506,and rebeccamycin); bulgarein; DNA minor groove binders such as Hoechstdye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; antisense oligonucleotides (e.g.,those disclosed in the U.S. Pat. Nos. 6,277,832, 5,998,596, 5,885,834,5,734,033, and 5,618,709, all of which are herein incorporated byreference in their entirety); adenosine deaminase inhibitors (e.g.,fludarabine phosphate and 2-chlorodeoxyadenosine); and pharmaceuticallyacceptable salts, solvates, clathrates, and prodrugs thereof.

In another embodiment, the targeting antibody, such as a CD20, CD22,bispecific CD1d/CD5, or bispecific CD24/CD27 antibody, can be conjugatedto a radioactive metal ion, such as the alpha-emitters ²¹¹astatine,²¹²bismuth, ²¹³bismuth; the beta-emitters ¹³¹iodine, ⁹⁰yttrium,¹⁷⁷lutetium, ¹⁵³samarium, and ¹⁰⁹palladium; or macrocyclic chelatorsuseful for conjugating radiometal ions, including but not limited to,¹³¹indium, ¹³¹L, ¹³¹yttrium, ¹³¹holmium, ¹³¹samarium, to polypeptides orany of those listed supra. In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid(DOTA), which can be attached to the antibody via a linker molecule.Such linker molecules are commonly known in the art and described inDenardo, et al., 1998, Clin Cancer Res 4(10):2483-90; Peterson, et al.,1999, Bioconjug Chem 10(4):553-7; and Zimmerman, et al., 1999, Nucl MedBiol 26(8):943-50, each incorporated by reference herein in theirentireties.

In still another embodiment, the targeting antibody, such as a CD20,CD22, bispecific CD1d/CD5, or bispecific CD24/CD27 antibody, can beconjugated to a proteinaccous agent that modifies a given biologicalresponse and leads to cytotoxicity. In one embodiment, the antibody isconjugated to a plant-, fungus-, or bacteria-derived toxin. Non-limitingexamples of such toxins include A chain toxins, ribosome inactivatingproteins, ricin A, deglycosylated ricin A chain, abrin, alpha sarcin,aspergillin, restrictocin, ribonucleases, diphtheria toxin, bacterialendotoxin, saporin toxin, Granzyme B or the lipid A moiety of bacterialendotoxin, cholera toxin, or Pseudomonas exotoxin and derivatives andvariants thereof.

In another embodiment, an antagonist capable of engaging a B10 cellspecific surface marker to ablate the B10 cell population is a syntheticligand specific for the marker, such as that described in Collins etal., 2006, J. Immunol. 5:2994-3003, incorporated herein by reference inits entirety. In one aspect of this embodiment, the synthetic ligand maybe further conjugated to a toxin, such as the saporin toxin.

In an alternative embodiment, a subject suffering from cancer, aninfectious disease or an immune deficiency disease associated withelevated levels of IL-10 is treated by administration of a compoundcapable of engaging a marker or markers on the B10 cell subset that caninhibit the production of IL-10 by the B10 cells. Non-limiting examplesof such compounds include antibodies and fragments thereof, smallmolecules, synthetic drugs, peptides (including cyclic peptides),polypeptides, proteins, nucleic acids, synthetic or natural inorganicmolecules, mimetic agents, and synthetic or natural organic molecules.In one embodiment, the compound engages CD22. In an aspect of thisembodiment, the compound is a CD22 antibody. In another aspect of thisembodiment, the compound engages CD5. In an aspect of this embodiment,the compound is a CD5 antibody. In another aspect of this embodiment,the compound engages CD1d. In an aspect of this embodiment, the compoundis a CD1d antibody. In still another aspect of this embodiment, thecompound is a bispecific CD1d/CD5 antibody. In another aspect of thisembodiment, the compound engages CD24. In an aspect of this embodiment,the compound is a CD24 antibody. In another aspect of this embodiment,the compound engages CD27. In an aspect of this embodiment, the compoundis a CD27 antibody. In still another aspect of this embodiment, thecompound is a bispecific CD24/CD27 antibody. In yet another aspect ofthis embodiment, the compound engages CD19. In an aspect of thisembodiment, the compound is a CD19 antibody.

An antibody according to these embodiments can be any type of antibodyor fragment thereof, as described above. According to this embodiment,administration of an antibody that targets the B10 cell population orfragment thereof, including a CD22 antibody or fragment thereof to apatient with cancer, an infectious disease, an autoimmune disease, or aninflammatory disease associated with increased levels of IL-10 resultsin a downegulation of IL-10 production by the B10 cell population in thepatient.

In another embodiment, a patient suffering from cancer, an infectiousdisease or an immune deficiency disease associated with elevated levelsof IL-10 is treated by administration of an antibody that binds to a Bcell marker and selectively depletes the B10 cell population in thepatient. According to this embodiment, the B cell marker can be anyantigen that is presently known or subsequently determined to beexpressed by B10 cells including, e.g. CD1d, CD5, CD19, CD20, CD21,CD23, CD24, CD25, CD27, CD3, CD48, and CD148. In one aspect of thisembodiment, the antibody that binds to a B cell marker and selectivelydepletes the B10 cell population in the patient does not cause depletionof the B10 cell population by an antibody-dependent cell-mediatedcytotoxicity (ADCC) mechanism, by complement-dependent cytotoxicity(CDC), or by apoptosis. In another aspect, depletion of the B10 cellpopulation by the antibody is independent of the antibody's Fc region.In another aspect of this embodiment, the antibody that binds to a Bcell marker and selectively depletes the B10 cell population depletessplenic Marginal Zone B cells but does not substantially deplete splenicFollicular B cells. In a specific aspect, the antibody that binds to a Bcell marker and selectively depletes the B10 cell population is an IgG2bor an IgG3 isotype.

In another embodiment, the antibody for use in treating a patientsuffering from cancer or an immune deficiency disease associated withelevated levels of IL-10 that binds to a B cell marker and selectivelydepletes the B10 cell population comprises a human IgG isotype or Fcregion that does not activate complement or lead to ADCC or kill cellsby inducing apoptosis. Any human isotype or Fc region that does notactivate complement or lead to ADCC or kill cells by inducing apoptosiscan be used in accordance with this embodiment. In one aspect, theisotype is IgG4.

In a specific embodiment, a patient suffering from cancer is treated byadministration of a CD20 antibody that selectively depletes the B10 cellpopulation in the patient, wherein the depletion of the B10 cellpopulation by the CD20 antibody is not caused by ADCC, CDC, orapoptosis. In another aspect, depletion of the B10 cell population bythe antibody is independent of the antibody's Fc region. In an aspect ofthis embodiment, the CD20 antibody depletes splenic Marginal Zone Bcells but does not substantially deplete splenic Follicular B cells. Ina specific aspect, the CD20 is an IgG2b or an IgG3 isotype. In anotheraspect, the CD20 antibody comprises a human IgG isotype or Fc regionthat does not activate complement or lead to ADCC or induce apoptosis.Any human isotype or Fc region that does not activate complement or leadto ADCC or kill cells by inducing apoptosis can be used in accordancewith this embodiment. In one aspect, the isotype is IgG4.

In certain embodiments, the B10 cell population is depleted by at least1%, at least 1% to 5%, at least 1% to 10a/6, at least 1% to 25%, atleast 1% to 50%, at least 55%, at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99%, or by 100% as measured by assays known to one of skill in theart including those described in the Examples infra, e.g.,immunofluorescence staining with flow cytometry analysis, ELISA assayfor IL-10 secretion, or ELISpot analysis for determining numbers ofIL-10-secreting cells.

In certain embodiments, the antibodies described herein are administeredalone. In other embodiments, the antibodies described herein areadministered to patients as a front-line therapy. In other embodiments,the antibodies described herein are administered to patients as asecondary therapy. In certain embodiments, the patient has notpreviously been treated for the cancer, infectious disease or the immunedeficiency disease. In other embodiments, the patient is undergoing orhas undergone treatment for the cancer or the immune deficiency disease.In yet other embodiments, the patient has failed treatment for thecancer or the immune deficiency disease.

In certain embodiments, the antibodies described herein are administeredin combination with other therapeutic agents. Any therapy that isuseful, has been used, or is currently being used for the prevention,treatment, and/or management of cancer, infectious disease or an immunedeficiency disease can be used in compositions and methods of theinvention. Such therapies include, but are not limited to, peptides,polypeptides, antibodies, conjugates, nucleic acid molecules, smallmolecules, mimetic agents, synthetic drugs, inorganic molecules, andorganic molecules.

Non-limiting examples of cancer therapies include chemotherapy,radiation therapy, hormonal therapy, surgery, small molecule therapy,anti-angiogenic therapy, differentiation therapy, epigenetic therapy,radioimmunotherapy, targeted therapy, and/or biological therapyincluding immunotherapy including, but not limited to acivicin;aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin;altretamine; ambomycin; ametantrone acetate; aminoglutethimide;amsacrine; anastrozole; anthracyclin; anthramycin; asparaginase;asperlin; azacitidine (Vidaza); azetepa; azotomycin; batimastat;benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate;bisphosphonates (e.g., pamidronate (Aredria), sodium clondronate(Bonefos), zoledronic acid (Zometa), alendronate (Fosamax), etidronate,ibandornate, cimadronate, risedromate, and tiludromate); bizelesin;bleomycin sulfate; brequinar sodium; bropirimine; busulfan;cactinomycin; calusterone; caracemide; carbetimer, carboplatin;carmustine; carubicin hydrochloride; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate;cyclophosphamide; cytarabine (Ara-C); dacarbazine; dactinomycin;daunorubicin hydrochloride; decitabine (Dacogen); demethylation agents,dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; EphA2 inhibitors; elsamitrucin; enloplatin; enpromate;epipropidine; epirubicin hydrochloride; erbulozole; esorubicinhydrochloride; estramustine; estramustine phosphate sodium; etanidazole;etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride;fazarabine; fenretinide; floxuridine; fludarabine phosphate;fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine;histone deacetylase inhibitors (HDACs) gemcitabine hydrochloride;hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; imatinibmesylate (Gleevec, Glivec); interleukin II (including recombinantinterleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b;interferon alpha-n1; interferon alpha-n3; interferon beta-I a;interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotideacetate; lenalidomide (Revlimid); letrozole; leuprolide acetate;liarozole hydrochloride; lometrexol sodium; lomustine; losoxantronehydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride;CD2 antibodies (e.g., siplizumab (MedImmune Inc.; InternationalPublication No. WO 02/098370, which is incorporated herein by referencein its entirety)); megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxaliplatin; oxisuran;paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride; 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor, bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor,carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, dioxamycin; diphenyl spiromustine; docetaxel; docosanol;dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene;emitefur; epirubicin; epristeride; estramustine analogue; estrogenagonists; estrogen antagonists; etanidazole; etoposide phosphate;exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; HMG CoAreductase inhibitors (e.g., atorvastatin, cerivastatin, fluvastatin,lescol, lupitor, lovastatin, rosuvastatin, and simvastatin); hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor, interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-iroplact;irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor, leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; LFA-3TIP(Biogen, Cambridge, MA; International Publication No. WO 93/0686 andU.S. Pat. No. 6,162,432); liarozole; linear polyamine analogue;lipophilic disaccharide peptide; lipophilic platinum compounds;lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine;losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinaseinhibitors; menogaril; merbarone; meterelin; methioninase;metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim;mismatched double stranded RNA; mitoguazone; mitolactol; mitomycinanalogues; mitonafide; mitotoxin fibroblast growth factor-saporin;mitoxantrone; mofarotene; molgramostim; monoclonal antibody, humanchorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wallsk; mopidamol; multiple drug resistance gene inhibitor, multiple tumorsuppressor 1-based therapy; mustard anticancer agent; mycaperoxide B;mycobacterial cell wall extract; myriaporone; N-acetyldinaline;N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine;napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronicacid; neutral endopeptidase; nilutamide; nisamycin; nitric oxidemodulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxelderivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor, platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator, protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor, retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor, stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;5-fluorouracil; leucovorin; tamoxifen methiodide; tauromustine;tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomeraseinhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide;tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene bichloride; topsentin; toremifene; totipotent stem cellfactor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; thalidomide; velaresol; veramine; verdins; verteporfin;vinorelbine; vinxaltine; VITAXIN™ (see U.S. Patent Pub. No. US2002/0168360 A1, dated Nov. 14, 2002, entitled “Methods of Preventing orTreating Inflammatory or Autoimmune Disorders by Administering Integrinαvβ3 Antagonists in Combination With Other Prophylactic or TherapeuticAgents”); vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer.

5.7 Vaccine Formulations

In another embodiment, a therapeutic agent capable of ablating the B10cell subset can be administered in conjunction with a vaccine or otherantigen in order to increase the immune response directed against aninfectious disease or cancer-associated target, e.g., a tumor orantigen. In the methods, the antigen is administered to the subject andan agent that kills or inhibits the function, localization or expansionof the IL-10 producing B10 cells or an agent that inhibits production ofIL-10 by B10 cells in subject is also administered to the subject. Theadministration of the antigen and the agent increases or enhances theimmune response directed to the antigen after administration as comparedto the immune response directed to the antigen if administered in theabsence of the agent. In particular, the method increases Ig classswitching and in particular the levels of IgG are enhanced. According tothis embodiment, ablation of the B10 cell subset serves to decreaseendogenous levels of IL-10 in the subject being vaccinated oradministered the antigen and thereby boosts the immune response, inparticular the IgG response, directed to the infectious agent, infectedcells, antigen or tumor antigen. Any antigen, including antigensassociated with any infectious disease or malignant cell can bevaccinated against according to this method of the invention.

A non-limiting list of FDA licensed vaccines (and associated disease)that could be administered in accordance with the methods of theinvention includes: Acel-Immune (Diphtheria, tetanus, pertussis), ActHIB(Haemophilus influenzae type b), Anthrax vaccine, Attenuvax (Measles),Biavax II (Rubella, Mumps), Botox (Botulism), Chickenpox vaccine,Cholera vaccine, Comvax (Haemophilus influenzae type b, Hepatitis B),DTP (Diphtheria, Tetanus, Pertussis), Diphtheria vaccine, Engerix-B(Hepatitis B), Influenza vaccine, Fluvirin (Influenza), German Measlesvaccine, Havrix (Hepatitis A), HBIG (Hepatitis B), Hepatitis A vaccine,Hepatitis B vaccine, Heptavax (Hepatitis B), HibTITER (Haemophilusinfluenzae type b, Diphtheria), Imovax Rabies vaccine, Infanrix(Diphtheria, Tetanus, Pertussis), Ipol (Polio), JE-Vax (JapaneseEncephalitis Virus), Pedvax-HIB (Haemophilus influenzae type b,Meningitis), Meningococcal polysaccharide vaccine (Meningitis),Menomune-A/C/Y/W-135 (Meningitis), Meruvax-II (Rubella), M-M-R II(Measles, Mumps, Rubella), M-R-VAX II (Measles, Mumps, Rubella),Mumpsvax (Mumps), OmniHIB (Haemophilus influenzae type b, Diphtheria),Orimune (Polio), Paratyphoid vaccine (Typhoid), Pertussis vaccine,Plague vaccine, Pneumococcal vaccine (Pneumonia), Pneumovax 23(Pneumonia), Pne-Imune 23 (Pneumonia), Polio vaccine, Recombivax HB(Hepatitis B), RhoGAM (Rhesus), Rocky Mountain Spotted Fever vaccine,Rubella vaccine, Rubeola vaccine, Smallpox vaccine, Tetanus vaccine,Tetramune (Diphtheria, Tetanus, Pertussis, Haemophilus influenzae typeb), Tice BCG USP (Mycobacterium Bovis Infection), Tri-Immunol(Diphtheria, Tetanus, Pertussis), Tripedia(Diphtheria, Tetanus,Pertussis), Typhim Vi (Typhoid), Typhoid vaccine, Typhus vaccine, Vaqta(Hepatitis A), Varicella vaccine, Varivax (Varicella), Vivotif Berna(Typhoid), and Yellow Fever vaccine. Some infectious diseases and tumorshave been resistant to immunization protocols or have demonstratedlimited immunity or required multiple boosts to elicit an effectiveimmune response. The methods described herein may be combined with knownvaccines or vaccines that failed during development stages to elicit arobust immune response to the antigen to reduce the number of boostersrequired or to increase the immune response directed to the antigen andincrease the efficacy of the vaccine. As an example, the Mycobacteriumbovis BCG vaccine is known to only provide partially protective immunityand thus is not used to vaccinate against tuberculosis in much of theworld. The BCG vaccine could be administered with a an agent thatinhibits the function, localization or expansion of B10 cells or anagent that inhibits IL-10 production to help elicit a more robust immuneresponse to the vaccine.

In one aspect of this embodiment, the therapeutic agent capable ofablating the B10 cell subset and the vaccine or antigen are administeredconcurrently. In another aspect of this embodiment, the therapeuticagent capable of ablating the B10 cell subset is administered prior toadministration of the vaccine or antigen. Alternatively, the therapeuticagent capable of ablating the B10 cell subset can be administeredfollowing the administration of the vaccine or antigen.

In another aspect of this embodiment, the therapeutic agent capable ofablating the B10 cell subset and the vaccine are administered inconjunction with an adjuvant. Anon-limiting list of adjuvantsadministered in accordance with the methods of the invention includes:alum (e.g., aluminum hydroxide, aluminum phosphate); Montanide ISA 720;MF-59; PROVAX; immunostimulatory nucleic acids, such as CpGoligodeoxynucleotides; saponins purified from the bark of the Q.saponaria tree, such as QS21; poly[di(carboxylatophen-oxy)phosphazene,derivatives of lipopolysaccharides (LPS), such as monophosphoryl lipidA, muramyl dipeptide (MDP; Ribi) andthreonyl-muramyl dipeptide (t-MDP;Ribi); OM-174; Leishmania elongation factor, ISCOMs; SB-AS2; SB-AS4;non-ionic block copolymers that form micelles such as CRL 1005; SyntexAdjuvant Formulation CpG nucleic acids; Bacterial toxins, e.g., Choleratoxin (CT), CT derivatives including but not limited to CT B subunit(CTB); Zonula occludens toxin, zot; Escherichia coli heat-labileenterotoxin; Labile Toxin (LT), LT derivatives including but not limitedto LT B subunit (LTB); Pertussis toxin, PT; toxin derivatives; Lipid Aderivatives (e.g., monophosphoryl lipid A, MPL); bacterial outermembrane proteins (e.g., outer surface protein A (OspA) lipoprotein ofBorrelia burgdorferi, outer membrane protein of Neisseria meningitidis).

In the methods described herein, the increase immune response inresponse to administration of the antigen and an agent that reduces B10cell function, localization expansion or production of IL-10 can bemeasured by methods known to those of skill in the art including but notlimited to ELISA, Western blot, ELISpot, dot blot. The enhanced immuneresponse may include enhanced antigen specific antibody production andin particular enhanced class switching, e.g. enhanced IgG production. Asdescribed above the subjects include mammals, including domesticatedanimals such as livestock, pets, and primates including monkeys andhumans.

5.8 Diagnostics

In another embodiment, methods are provided for diagnosing a subjectsuffering from a disease that is associated with elevated or diminishedlevels of IL-10 production. In another embodiment, a subject with apredisposition to a certain disease can be diagnosed. In an aspect ofthese embodiments, B10 cells are isolated from the subject and assayedfor specificity to a certain disease-specific antigen.

The B10 cells to be analyzed may be collected from any location in whichthey reside in the subject including, but not limited to, blood, spleen,thymus, lymph nodes, and bone marrow. The isolated B10 cells may beanalyzed intact, or lysates may be prepared for analysis.

Methods for the quantitation of cells and detection of antigenicspecificity are known in the art, and may include pre-labeling thesample directly or indirectly; adding a second stage antibody that bindsto the antibodies or to an indirect label, e.g., labeled goat anti-humanserum, rat anti-mouse, and the like. For example, see U.S. Pat. No.5,635,363. Generally, assays will include various negative and positivecontrols, as known in the art.

Various methods are used to determine the antigenic specificity profilefrom a patient sample. The comparison of a binding pattern obtained froma patient sample and a binding pattern obtained from a control, orreference, sample is accomplished by the use of suitable deductionprotocols including, but not limited to, AI systems, statisticalcomparisons, and pattern recognition algorithms. Typically a data matrixis generated, where each point of the data matrix corresponds to areadout from a specific epitope. The information from reference patternscan be used in analytical methods to determine relative abundance,changes over time, and any other factors relevant to analysis.

Any disease can be diagnosed according to these embodiments. Inparticular, diseases associated with diminished levels of endogenousIL-10, e.g., immune and inflammatory diseases, and diseases associatedwith elevated levels of endogenous IL-10, e.g., cancer can be diagnosedbased on isolation of B10 cells in a subject with disease-specificantigen specificity.

In another embodiment, a subject diagnosed with a given disease can bemonitored for disease progression. Formats for patient sampling includetime courses that follow the progression of disease, comparisons ofdifferent patients at similar disease stages, e.g., early onset, acutestages, recovery stages; and tracking a patient during the course ofresponse to therapy. In an aspect of this embodiment, the numbers of B10cells having specificity to a certain disease-specific antigen can bemonitored over the course of a given therapy. As a non-limiting example,a therapy designed to expand the endogenous population of B10 cells thatrespond to a given disease should result in an increase in the numbersof B10 cells with specificity to a certain antigen associated with saiddisease relative to the general population of B10 cells.

6. Example 1: CD22 Antibodies Deplete the Regulatory B Cell Populationand Depletion Enhances Igg Production

Administration of CD22 mAbs to mice results in depletion of theregulatory B cell population as evidenced by a decrease inCD1d^(high)CD5⁺ B cells (FIG. 1A) and a decrease in B cell IL-10production (FIG. 1B). Thus, CD22 mAb treatment efficiently depletedspleen B10 cells while leaving most other spleen B cells intact.Effective B10 cell depletion by MB22-10 mAb in wild-type mice provides asystem to investigate whether B10 cell depletion by CD22 mAb wouldenhance the IgG responses in wild-type mice immunized with a T celldependent antigen in the absence of adjuvant. DNP-KLH immunizationinduced significant primary and secondary (day 21 boost) DNP-specificIgM responses in control mAB-treated wild-type mice, while DNP-specificIgG responses rose only slightly above background levels (FIG. 1C). Bycontrast, CD22 mAb-treated mice generated normal Ag-specific IgMresponses and robust DNP-specific IgG1, IgG2c, IgG2b, and IgG3 Abresponses that remained high. CD22 mAb treatment also significantlyexpanded the frequency of spleen IgG-secreting B cells in comparisonwith control mAb-treated mice (FIG. 1D). Thereby B10 cell depletiondramatically enhances IgG responses in wild-type mice.

7. Example 2: Characterization of the Human B19 Cell Population

The results shown below identify IL-10 competent B10 and B10pro cells inman that are comparable to those identified in mice, and show that bothadaptive and innate signals can drive human B10 cell maturation andIL-10 production.

7.1 Materials and Methods 7.1.1 Cell Preparation

Heparinized blood samples were obtained from healthy 22 to 53 year-oldadult donors or from patients with autoimmune disease, with B cellsexamined immediately thereafter. Tissue samples were obtainedanonymously at surgery or postmortem from individuals withoutidentifiable hematologic disorders, with the B cells purified andimmediately cryopreserved and then kept frozen in liquid nitrogen untiluse. Tonsils were obtained from patients undergoing a routinetonsillectomy. Umbilical cord blood samples were obtained from frozenresearch units that were processed at the Duke University Stem CellLaboratory and the Carolinas Cord Blood Bank.

7.1.2 Mice

C57BL/6 mice were from the Jackson Laboratory (Bar Harbor, ME). All micewere housed in a specific pathogen-free barrier facility and used at8-12 wk of age.

7.13 Antibodies

Anti-human mAbs included: IgD (IA6-2) from BD PharMingen (San Diego,CA); CD21 (BU33), CD22 (RFB4), CD23 (D.6) from Ancell (Bayport, MN); IgM(MHM-88), CD1d (51.1), CD5 (UCHT2), CD19 (HIB19), CD24 (ML5), CD25(BC96), CD27 (0323), CD38 (HIT2), CD40 (HB14), CD48 (BJ40), and CD148(A3) mAbs from BioLegend (San Diego, CA). Anti-human IgM Ab was fromJackson ImmunoResearch Laboratories, Inc. (West Grove, PA).Phycoerythrin-conjugated anti-human IL-10 mAb (JES3-19F1) was fromBioLegend (San Diego, CA).

Anti-mouse mAbs included: CD20 mAb (MB20-11; Uchida et al., 2004. Int.Immunol. 16:119-129); B220 mAb RA3-6B2 (obtained from Dr. RobertCoffman, DNAX Corp., Palo, Alto, CA); and CD19 (1D3), CD5 (53-7.3), CD1d(1B1), CD21/35 (7G6), CD23 (B3B4), CD24 (Ml/69), CD25 (PC61), and CD40(3/23) mAbs from BD PharMingen (San Diego, CA); CD27 (LG.3A10), CD38(90) from BioLegend (San Diego, CA); IgM (11/41) from eBioscience (SanDiego, CA); and IgD (11-26) from Southern Biotechnology Associates(Birmingham, AL); FITC-conjugated anti-mouse CD22 N-terminus (Cy34,T1B163, American Type Culture Collection). Phycoerythrin-conjugatedanti-mouse IL-10 mAb (JES5-16E3) was from eBioscience (San Diego, CA).

7.1.4. B Cell Isolation, Immunofluorescence Analysis and Cell Sorting

Blood mononuclear cells were isolated from heparinized blood aftercentrifugation over a discontinuous Lymphoprep (Axis-Shield PoC As,Oslo, Norway) gradient. Single cell splenocyte, lymph node, and tonsilsuspensions were generated by gentle dissection with >90% cell viabilityas determined by trypan blue exclusion. Cell numbers were quantifiedusing a hemocytometer, with relative lymphocyte percentages among viablecells (based on scatter properties) determined by flow cytometryanalysis. In some experiments, B cells were enriched using RosetteSep(STEMCELL Technologies, Vancouver, BC, Canada) following themanufacturer's protocols. CD19-mAb coated microbeads (Miltenyi Biotech)were used to purify blood B cells by positive selection following themanufacturer's instructions. When necessary, the cells were enriched asecond time using a fresh MACS column to obtain>99% purities.

Single cell leukocyte suspensions were stained on ice usingpredetermined optimal concentrations of each Ab for 20-60 min, and fixedas described (Sato et al., 1996, J. Immunol. 157:4371-4378). Cells withthe light scatter properties of lymphocytes were analyzed by 2-6 colorimmunofluorescence staining and FACScan or FACSCalibur flow cytometers(Becton Dickinson, San Jose, CA). Dead cells were excluded from theanalysis based on their forward- and side-light scatter properties andthe use of LIVE/DEAD Fixable Dead Cell Stain Kits (Invitrogen-MolecularProbes, Carlsbad, CA). All histograms are shown on a 4-decadelogarithmic scale, with gates shown to indicate backgroundisotype-matched control mAb staining set with <2% of the cells beingpositive. Blood CD24^(hi)CD27+ and CD24^(low)CD27⁻ B cells were isolatedusing a FACSVantage SE flow cytometer (Becton Dickinson, San Jose, CA)with 90-95% purities.

7.1.5. Analysis of IL-10 Production

Intracellular IL-10 analysis by flow cytometry was as described (Yanabaet al., 2008, Immunity 28:639-650). Briefly, isolated mononuclear cellsor purified B cells were resuspended (2×10⁶ cells/ml) in complete medium[RPMI 1640 media containing 10% FCS, 200 μg/ml penicillin, 200 U/mlstreptomycin, and 4 mM L-Glutamine, with 5×10⁻⁵ M 2-mercaptoethanol inmice, without 2-mercaptoethanol in human (all from Gibco, Carlsbad,CA)]. The cells were stimulated with LPS (10 μg/ml, Escherichia coliserotype 0111: B4; Sigma), CpG (human ODN 2006, mouse ODN 1826, 10μg/ml; Invivogen), or other TLR agonists (TLR1, Pam3CSK4, 1 μg/ml; TLR2,heat-killed Listeria monocytogenes, 10⁸ cells/ml; TLR3, Poly(I:C), 10μg/ml; TLR5, S. typhimurium flagellin, 1 μg/ml; TLR6, Pam2CGDPKHPKSF, 1μg/ml; TLR7, Imiquimod, 1 μg/ml; TLR8, ssRNA40, 1 μg/ml; all fromInvivogen), CD40L (1 μg/ml; R&D Systems, Minneapolis, MN), anti-humanCD40 mAb (1 μg/ml; BioLegend), anti-mouse CD40 mAb (1 μg/ml; BDPharmingen), PMA (50 ng/ml; Sigma), ionomycin (human 1 μg/ml, mouse 500ng/ml; Sigma), BFA (1X solution/ml; BioLegend), monensin (2 mM;eBioscience), and anti-human IgM Ab (10 μg/ml; Jackson ImmunoResearchLaboratories) as indicated for 5 or 48 h, in 48-well flat-bottom platesbefore staining and flow cytometry analysis. For analysis of cellproliferation, lymphocytes were stained with CFSE Vybran™ CFDA SEfluorescent dye (5 μM; Invitrogen-Molecular Probes) according to themanufacturer's instructions. For IL-10 detection, Fc receptors wereblocked with mouse Fc receptor mAb (2.4G2; BD PharMingen) or humanFcγR-Binding inhibitor (eBioscience) with dead cells detected by using aLIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen-MolecularProbes) before cell surface staining. Stained cells were fixed andpermeabilized using a Cytofix/Cytoperm kit (BD PharMingen) according tothe manufacturer's instructions and stained withphycoerythrin-conjugated anti-human or anti-mouse IL-10 mAb.

Secreted IL-10 was quantified by ELISA. Purified B cells (4×10⁵) werecultured in 0.2 ml of complete medium in a 96-well flat-bottom tissueculture plates. Culture supernatant fluid IL-10 concentrations fortriplicate samples were quantified using IL-10 OptEIA ELISA kits (BDPharMingen) following the manufacturer's protocols.

7.1.6. B Cell IL10 Transcript Expression

In some experiments, IL-10-secreting blood B cells were identified after4 h of in vitro stimulation using an IL-10 secretion detection kit(Miltenyi Biotech, Auburn, CA) with subsequent staining for CD19expression before cell sorting into IL-10⁺ CD19⁺ and IL-10⁻ CD19⁺populations. Total RNA was extracted from the purified B cells usingQiagen RNeasy spin columns (Qiagen Ltd., Crawley, UK). Random hexamerprimers (Promega, Madison, WI) and Superscript II RNase H ReverseTranscriptase (Invitrogen, Carlsbad, CA) were used to generate cDNA.IL-10 transcripts were quantified by real-time PCR analysis using SYBRGreen as the detection agent as described. The PCR was performed withthe iCycler iQ system (Bio-Rad, Hercules, CA). All components of the PCRmix were purchased from Bio-Rad and used according to the manufacturerinstructions. The reaction conditions were as follows: 2 min at 50° C.(1 cycle), 10 min at 95° C. (1 cycle), 15 s at 95° C., and 1 min at 60°C. (50 cycles). Specificity of the RT-PCR was controlled by thegeneration of melting curves. Relative expression of PCR products wasdetermined using the ΔΔCT method. Briefly, each set of samples wasnormalized using the difference in threshold cycle (CT) between thetarget gene and housekeeping gene (GAPDH): ΔCT=(CT target gene−CTGAPDH). Relative mRNA levels were calculated by the expression2^(−ΔΔCT), where ΔΔCT=ΔCT sample−ΔCT calibrator. For all reactions, eachcondition was performed in triplicate. Data analysis was performed usingiQ Cycler analysis software. The sense IL-10 primer was5′-CTTCGAGATCTCCGAGATGCCTTC-3′ (SEQ ID NO: 6) and the antisense primerwas 5′-ATTCTTCACCTGCTCCACGGCCTT-3′ (SEQ ID NO: 7). The sense GAPDHprimer was 5′-GCCACCCAGAAGACTOTGGATGGC-3′ (SEQ ID NO: 8) and theantisense primer was 5′-CATGTAGGCCATGAGGTCCACCAC-3′ (SEQ ID NO: 9).

7.1.7. Patients

All subjects with rheumatoid arthritis (RA) met the American College ofRheumatology 1987 revised criteria for classification (Arnett et al.,1988, Arthritis Rheum. 31:315-324); subjects with systemic lupuserythematosus (SLE) satisfied the 1982 revised criteria for theclassification (Tan et al., 1982, Arthritis Rheum. 25:1271-1277); andsubjects with primary Sjögren's syndrome (SjS) fulfilled theAmerican-European consensus group revised criteria for theclassification (Vitali et al., 2002, Ann. Rheum. Dis. 61:554-558).Patients with autoimmune vesiculobullous skin disease (BD) includedbullous pemphigoid (BP), pemphigus foliaceus (PF), and pemphigusvulgaris (PV). All patients had typical clinical and histologic findingswith diagnostic findings on direct immunofluroesence of perilesionalskin or oral mucosa (Yancey and Egan, 2000, J. Amer. Med Assoc.284:350-356; Udey and Stanley, 1999, JAMA 282:572-576). Informed consentfor multiple sclerosis (MS) patient's blood samples was obtained in eachinstance according to protocols approved by the Institutional ReviewBoard of St. Luke's-Roosevelt Hospital Center Institute for HealthSciences. All patients with MS fulfilled 2005 revised McDonald criteriafor relapsing remitting or primary progressive MS (Polman et al., 2005,Ann. Neurol. 58:840-846). Secondary progressive MS was defined using theLublin and Reingold criteria (Lublin and Reingold, 1996, Neurology46:907-911). Most patients were receiving treatment withimmunomodulatory drugs and/or low doses of prednisone (see Table 1,below).

7.1.8. Statistical Analysis

All data are shown as means (±SEM). Significant differences betweensample means were determined using the Student's t test.

7.2 Results 7.2.1. Identification of Human IL-10-Producing B Cells

Mouse blood B10 cell frequencies were determined after culturing thecells with media, PIM, L+PIM, or CpG oligonucleotides plus PIM (CpG+PIM)for 5 h in vitro. Blood B cells did not express detectable IL-10 withoutin vitro stimulation. However, IL-10 competent B10 cells represented <3%of mouse blood B cells, with the combination of CpG+PIM inducing higherfrequencies of B10 cells than L+PIM or PIM alone (FIG. 2A). A similarstrategy to that used in mice was optimized to maximize human IL-10competent B10 cell enumeration. Brefeldin A (BFA) was used to blockIL-10 golgi transport rather than monensin since it optimized human Bcell cytoplasmic IL-10 expression (FIG. 2B). Optimal human B10 cellnumbers were observed after 5 h of PIB stimulation in vitro, withoverall B cell viability decreasing after this time point. BackgroundIL-10 mAb staining was reduced by the exclusion of all cell doublets anddead cells from the flow cytometry analysis with BFA cultures used asnegative controls (FIG. 2C). These assay conditions were then used toidentify human IL-10-competent blood B cells.

Human blood was found to contain a rare, but distinct subset ofIL-10-competent B10 cells that was detectable at low 0.25-2% frequenciesafter in vitro stimulation. B cell activation with PMA, ionomycin, andBFA (PIB) for 5 h induced 0.8±0.1% of B cells on average to expressIL-10 (n=14, 1.9±0.3×10⁻³B10 cells/ml, FIG. 2D-E). Some blood B cellsmay spontaneously express IL-10, but their frequencies and levels ofIL-10 expression were below the 0.2% threshold of quantification byimmunofluorescence staining; which is similar to background cytoplasmicIL-10 staining when using B cells from IL-10^(−/−) mice (see Yanaba etal., 2008, Immunity 28:639-650; Matsushita et al., 2008, J. Clin.Invest. 118:3420-3430; and Yanaba et al., 2009, J. Immunol.182:7459-7472). B cell stimulation using TLR agonists did notsubstantially alter mean B10 cell numbers, although IL-10⁺ B cellfrequencies were enhanced in some individuals by adding either CpG (TLR9agonist) or LPS to the PIB cultures. Thus, blood B10 cells were rare, atrait shared by both healthy humans and mice.

7.2.2. Human B10pro Cell Identification

In mice, B10pro cell maturation into IL-10-competent B10 cells can beinduced by 48 h stimulation with either LPS or agonistic CD40 mAb(Yanaba et al., 2009, J Immunol. 182:7459-7472). B10pro cells capable ofmaturing into IL-10 competent B10 cells after in vitro culture for 48 hwere identified in human blood by their ability to express cytoplasmicIL-10 after 5 h PIB stimulation (FIG. 2D). The total frequency of B10and B10pro cells (B10+B10pro) is quantified in this assay, as the Bcells that acquire IL-10 competence in vitro (e.g. matured B10pro cells)cannot be differentiated from preexisting blood B10 cells. Culturinghuman B cells with LPS, CpG, or recombinant CD40 ligand (CD40L, CD154)alone, together, or in combination with BFA did not induce detectablecytoplasmic IL-10 expression, while ˜0.2% of B cells cultured in mediaalone with 5 h PIB stimulation during the last 5 h of culture expressedcytoplasmic IL-10 (FIG. 2F-G). However, 1-4% of human blood B cellsexpressed IL-10 following TLR agonist (48 h)+PIB (5 h) stimulation.B10+B10pro cell frequencies increased to 0.6±0.1, 1.9±4.4, 0.8±1,1.2±0.2, and 4.1±1.0% following TLR1 agonist, LPS, TLR6 agonist, TLR7agonist, and CpG stimulation, respectively.

The addition of recombinant CD40L alone to B cell cultures did notinduce B10pro cell maturation, while the addition of CD40L to B cellsstimulated with LPS induced higher frequencies of B10+B10pro cells.CD40L induced 47% higher frequencies of IL-10⁺ B10 cells than agonisticCD40 mAb (FIG. 2F). CD40L stimulation also significantly enhanced meanB10+B10pro cell frequencies when combined with TLR1 agonist (1.1±0.2%),LPS (3.4±0.7%), TLR6 agonist (1.4±0.2%), TLR7 agonist (2.2±0.4%), or CpG(7.0±1.4%) stimulation (FIG. 2G right panel). Thus, dual CD40 and TLRstimulation induced the highest frequencies of human B10pro cells tobecome IL-10 competent B10 cells, with the highest numbers of B10+B10procells (1.6±0.3×10⁴ cells/ml, n=14) induced after 48 h of CD40L plus CpGstimulation. Thereby, human blood B10 and B10pro cells can be readilyand reproducibly quantified after stimulation or in vitro maturationwith CD40 ligation in combination with select TLR signals.

7.2.3. B10 Cell Numbers in Newborn Blood and Adult Lymphoid Tissues

Newborn mice have higher spleen B10 and B10pro cell frequencies thanadult mice (Yanaba et al., 2009, J Immunol. 182:7459-7472). Mean B10cell frequencies in human newborn blood after 5 h of CpG+PIB stimulation(0.45±0.14%, n=8; FIG. 3A) were 42% lower than those observed for adulthuman blood (FIG. 2E), although this may reflect donor pool diversityrather than represent differences between newborns and adults.Nonetheless, B10+B10pro cell frequencies were similar or higher innewborn blood relative to adult human blood after culture with CD40L andTLR agonists; TLR1 (2.6±0.6%), LPS (7.6+1.8%), TLR6 (4.21.4%), or TLR9(CpG, 9.6±2.3%) agonists with PIB added during the final 5 h of culture.Thus, newborn and adult blood contained both B10 and B10pro cells.

B10 cells were also found within spleens (0.31+0.06, n-4, CpG+PIB) andtonsils (0.31±0.11, n=3, CpG+PIB) of individuals without known disease(FIG. 3B-C). Stimulating spleen and tonsil B cells with LPS or CpG incombination with CD40L also induced B10pro cells to mature into IL-10competent B10 cells, with B10+B10pro cell frequencies ranging from <0.5%to almost 30% (FIG. 3B). Human tonsil and spleen B10 cell frequencieswere numerically similar to those observed in blood, but spleenB10+B10pro cell frequencies were 2.2-fold higher. In these samples,human spleen and tonsil B10 cell frequencies were 62-85% lower thanthose observed for mouse spleen (0.3% vs. 2.1%) and lymph node (0.3% vs.0.8%), respectively, but B10+B10pro cell frequencies were higher withinhuman tissues. It was observed that CpG stimulation induced human B10procell maturation, while CpG failed to induce mouse B10pro cell maturation(FIG. 3C). Regardless, B10 cells represented a small subset of humanspleen and lymph node B cells.

7.2.4. Regulation of B10 Cell IL-10-Production and Secretion In Vitro

The time course of B10 cell IL-10 induction was assessed in vitro byquantifying IL-10 transcripts in cultured human blood B cells stimulatedwith CD40L+CpG. By 12, 24, and 48 h, B cell stimulation induced 6.8-,24-, and 5.9-fold higher II10 transcript levels, respectively, than wasobserved for unstimulated B cells (p<0.05; FIG. 4A). Human blood B10cells that were actively secreting IL-10 expressed 110 transcripts at19-fold higher levels than IL-10⁻ B cells after in vitro stimulation(FIG. 4B). Thus, B10 cell IL-10 expression paralleled II10 genetranscription.

The response of human B10+B10pro cells to CD40L, CpG, and antigenreceptor generated signals was examined. In comparison with CD40L alone,CpG alone induced the highest levels of B10pro cell maturation intoIL-10-competent B10 cells, which was further increased when both CD40Land CpG were added to the cultures (FIG. 4C). By contrast, BCR ligationusing mitogenic anti-IgM Ab did not induce cytoplasmic IL-10 expression,but actually inhibited the B10 cell inducing effects of CpG+CD40Lstimulation. In vitro BCR signals also inhibit mouse B10pro cellmaturation and cytoplasmic IL-10 induction (Yanaba et al., 2009, J.Immunol. 182:7459-7472). Among TLR agonists, LPS and CpG were also themost potent stimuli for inducing IL-10 secretion by human blood B cells(FIG. 4D) and mouse blood B cells (FIG. 4E). Thus, similar signalsregulate human and mouse B10 and B10pro cells to mature and expresscytoplasmic IL-10 in vitro.

7.2.5. Phenotypic Characterization of Blood and Spleen IL-10-Competent BCells

Whether human B10 cells represent a phenotypically defined B cell subsetwas determined by immunofluorescence staining. B cells that were eitheruntreated, stimulated with PIB, L+PIB, or CpG+PIB for 5 h, and/orpermeabilized, were found to express identical cell surface IgM, IgD,CD1d, CD5, CD10, CD19, CD21, CD22, CD23, CD24, CD25, CD27, CD38, andCD40 densities. The transport of newly synthesized proteins to the cellsurface is also inhibited by BFA. The use of ten-fold higher PMAconcentrations did not alter B cell surface phenotypes or survivalduring these 5 h assays, while the use of ionomycin at ten-fold higherconcentrations significantly altered B cell surface phenotypes even inthe presence of BFA due to extensive cell death. Since these cellsurface molecules were not affected by the stimulation and/or cellpermeabilization protocols used to visualize cytoplasmic IL-10expression, they were used to categorize the phenotype of freshlyisolated blood B10 cells. Half of blood B10 cells expressed high IgMlevels and low IgD levels (FIG. 5A). Both CD24 and CD27 expression werehigh on the majority of B10 cells, while IL-10⁻ B cells expressed eitherhigh or low density CD24 and CD27. CD19 and CD25 expression were alsohigher on B10 cells than IL-10⁻ B cells. Otherwise, the remaining cellsurface markers were absent or expressed similarly by both B10 cells andIL-10⁻ B cells. The same results were obtained following PIB, L+PIB, orCpG+PIB stimulation. Thereby, freshly isolated human bloodIL-10-competent B10 cells were predominantly CD24^(hi)CD27⁺ B cells.

The phenotype of blood B10+B10pro cells induced during 48 h in vitrocultures was also assessed. In comparison with freshly isolated B cellsafter 5 h of L+PIB stimulation (FIG. 5A), prolonged cell culture andthese stimulation conditions induced significant changes in the cellsurface phenotype of B10 and non-B10 cells (FIG. 5B). For example, mostB cells were induced to express CD25 and CD38 at high densities.Nonetheless, B10+B10pro cells on average expressed higher densities ofCD1d, CD19, CD20, CD21, CD23, CD24, CD25, CD27, and CD38 when comparedwith IL-10⁻ B cells, consistent with an activated phenotype. Spleen B10cells were also predominantly CD27⁺, although the expression of mostcell surface molecules was similar if not identical for B10 cells andIL-10′ B cells (FIG. 5C). Spleen B10+B10pro cells and IL-10⁻ B cellsalso had similar phenotypes after 48 h of stimulation in vitro, with theexception that IL-10⁺ B10 cells were predominantly IgM while IL-10⁻ Bcells were predominantly IgM^(low) (FIG. 5D). Thereby, spleen B10 cellswere also predominantly CD24^(hi)CD27⁺.

The phenotype of mouse blood B10 cells was also assessed for comparison.Mouse blood B10 cells expressed higher levels of IgM than IL-10′ Bcells, but most other cell surface molecules were expressed at similardensities (FIG. 5E). CD1d, CD5, CD19, CD24, and CD38 expression wereslightly higher on IL-10⁺ than IL-10⁻ B cells, while CD23 expression washeterogeneous in comparison to IL-10⁻ B cells. Mouse blood B10+B10procells were predominantly IgM^(hi)CD5⁺CD19^(hi)CD23^(low)CD24^(hi)CD38^(hi) after in vitro activation(FIG. 5F). Spleen B10 cells were predominantly IgM^(hi) IgD^(low)CD1d^(hi) CD5⁺CD19^(hi) CD21^(hi/int) CD23^(low) CD24^(hi) CD38^(hi)(FIG. 5G). Mouse spleen B10+B10pro cells were more similar to IL-10⁻ Bcells after 48 h of culture (FIG. 5H). Thereby, human blood B10 cellswere predominantly CD24^(hi)CD27⁺, while mouse spleen B10+B10pro cellswere predominantly CD1d^(hi)CD5⁺.

7.2.6. Blood B10 Cells are Enriched within the CD24^(hi)CD27⁺ B CellSubpopulation

When the spectrum of normal control blood donors was compared, blood B10cells were predominantly CD24^(hi) and CD27⁺ (FIG. 5A). Most B10 cellsalso expressed additional cell surface markers of activation (CD48^(hi))and memory (CD148^(hi)) (FIG. 6A). Since cell surface CD24, CD27, CD38,and CD48, and CD148 expression were not affected by the 5 h cultureconditions used to induce B cell cytoplasmic IL-10 expression (FIG. 6B),circulating B10 cells were predominantly CD24^(hi), CD27⁺, CD48^(hi),and CD148^(hi). Cell surface CD27 and CD38 expression profiles have beenused frequently to define human blood B cell subsets (see, e.g.,Levesque and St. Clair, 2008, J. Allergy Clin. Immunol. 121:13-21; andSanz et al., 2008, Sem. Immunol. 20:67-82). However, when blood B10cells were analyzed based on their CD38 versus IgD expression profiles,IL-10⁺B10 cells from representative blood donors fell into both theCD38^(hi) and CD38^(lo) populations (FIG. 6C). Similarly, when blood B10cells were analyzed based on CD27 versus IgD expression, IL-10⁺ B cellsfrom representative blood donors did not fall into pre-defined subsets,but were predominantly CD27^(hi).

To determine whether B10 cells predominantly localize within theCD24^(hi)CD27⁺ subpopulation that represented 24±5% (n=7) of blood Bcells, blood CD24^(hi)CD27⁺ and CD24^(low)CD27⁻ subpopulations werefirst purified and then cultured individually with L+PIB for 5 h toinduce IL-10 expression (FIG. 6D). Following cell permeabilization andcytoplasmic IL-10 staining, B10 cell frequencies were at least 10-foldhigher within the previously purified CD24^(hi)CD27⁺ subpopulation whencompared with the isolated CD24^(low)CD27⁻ B cells. Thus, B10 cells werepredominantly CD24^(hi)CD27⁺ in vivo.

To determine whether B10pro cells also predominantly localize within theCD24^(hi)CD27⁺ subpopulation, blood CD24^(hi)CD27⁺ and CD24^(low)CD27⁻subpopulations were purified and then cultured individually with CD40L,and LPS or CpG for 48 h to induce B10pro cell maturation. Again, thefrequency of B10 cells was 10-fold higher within the CD24^(hi) CD27⁺subpopulation when compared with CD24^(low)CD27⁻ cells (FIG. 6E). Toeliminate the possibility that B10pro cell proliferation during the 48 hcultures contributed to their expansion, the purified blood B cells werelabeled with CFSE before in vitro stimulation. Regardless of whether thecells were stimulated with LPS or CpG, there was no B cell divisionuntil 72-96 h of culture. At this point, B10 cells exhibited asignificant proliferative capacity, while IL-10⁻ B cells exhibited amodest proliferative capacity (FIG. 6F). Thus, the preferentiallocalization of blood B10 and B10+B10pro cells within the CD24^(hi)CD27⁺B cell subpopulation was not due to cell proliferation during the 48 hculture period.

The capacity of freshly isolated CD24^(hi)CD27⁺ and CD24^(low)CD27⁻ Bcells to secrete IL-10 was also assessed. CD40L, LPS, and LPS+CD40Lstimulation for 72 h induced modestly increased IL-10 production by theCD24^(hi)CD27⁺ population, but not CD24^(low) CD27⁺ cells (FIG. 6G).CD24^(hi)CD27⁺ cells secreted >10-fold more IL-10 in response to eitherCpG or CpG+CD40L stimulation when compared with LPS stimulation alone.IL-10 secretion by CD24^(low)CD27⁻ B cells was 78% and 82% lower thanfor CD24^(hi)CD27⁺ B cells in response to CpG and CpG+CD40L,respectively (p<0.001). Thus, blood B10 and B10+B10pro cells werepredominantly a small subset of the CD24^(hi)CD27⁺ B cell subpopulation.

7.2.7. B10 Cell Development Ln Patients with Autoimmune Disease

To determine whether blood B10 cell numbers are altered in patients withautoimmune disease, B10 and B10pro cells were examined in healthycontrols, and fifty-two patients with SLE, RA, SjS, autoimmune BD, orMS. Most of the patients were undergoing active treatment with immunemodulatory agents when the blood was taken (see Table 1, below).Nonetheless, some patients had high blood B10 cell frequencies eventhough there were no significant differences in mean B10 cell numbersbetween patient groups in comparison with controls (FIG. 7A-B). One BDpatient that had not been given immunosuppressive therapy had blood B10cell frequencies that were significantly higher than the populationmean. Two SLE patients and one RA patient also had blood B10 cellfrequencies that were significantly higher than the population mean, butretrospective evaluation of their disease status, autoantibody profile,and treatment regimen did not reveal obvious explanations for why theseindividuals had higher blood B10 frequencies than other patients. B10cell frequencies were similar regardless of whether LPS or CPG wereadded to the PIB-stimulated cultures (FIG. 7D, left panel). No patientgroups were identified that expressed significantly lower B10 cellfrequencies relative to controls or other patient groups.

Mean B10+B10pro cell frequencies from patients with SLE and BD weresignificantly higher than controls following CD40L+LPS stimulation,while mean B10+B10pro cell numbers in RA patient's were significantlyhigher after CD40L+CpG stimulation when compared with the control group(FIG. 7B). Notably, patients with high blood B10 cell frequencies didnot necessarily have high B10+B10pro cell frequencies after either LPSor CpG stimulation (FIG. 7C). While B10 cell frequencies were linearlycorrelated following either LPS or CPG stimulation, the scatter of theresults obtained for B10+B10pro cells was broad, suggesting inherentlydifferent patient sensitivities to LPS and CpG stimulation (FIG. 7D).Likewise, B10 and B10+B10pro cell frequencies did not correlate withCD27+ B cell frequencies. Regardless, relative B10 and B10⁺B10pro cellfrequencies were significantly correlated with the intensity ofcytoplasmic IL-10 expression (FIG. 7E). One patient among the entiregroup appeared to generate significantly higher (p<0.0 5) cytoplasmicIL-10 expression levels on a per cell basis relative to controls andother patients.

TABLE 1 Patient characteristics. Diagnosis Disease ImmunosuppressiveNumber Sex Age Duration (y) Autoantibody/Clinical Status Therapy RA01¹ M54 11 RF = 600 IU/ml; anti-CCP = 68.5 U/ml MTX, ADA, Pred 5 mg/d RA02 F44 4 RF = 352 IU/ml; anti-CCP > 100 U/ml MTX RA03 F 54 14 RF = 146 IU/mLMTX, LEF RA04 F 85 18 RF = neg MTX, IFX RA05 M 69 19 RF = 208 IU/ml;anti-CCP > 100 U/ml MTX, Pred 5 mg/d RA06 F 71 8 RF = neg MTX RA07 F 6713 RF = 333 IU/ml ETN RA08 F 58 25 RF = 53 IU/ml MTX, ETN, Pred 3 mg/dRA09 F 68 7 RF = 339 IU/ml MTX, Pred 3 mg/d RA10 M 75 13 RF = 420 IU/ml;anti-CCP = 18.9 U/ml MTX, LEF, Pred 10 mg/d RA11 F 73 7 RF and anti-CCP= neg MTX, LEF RA12 M 61 27 RF = 107 IU/ml MTX, LEF RA13 F 66 6 RF = posADA RA14 F 84 11 RF = 275 IU/ml; anti-CCP = 28 U/ml ETN RA15 F 52 3 RF =neg; anti-CCP > 100 U/ml ETN RA16 F 76 18 RF = pos MTX RA17 M 63 11 RF =neg; anti-CCP > 100 U/ml MTX, SSZ, Pred 1 mg/d RA18 F 43 15 RF = 23IU/ml MTX, IFX RA19 F 62 30 RF = 148 IU/ml; anti-CCP > 100 U/ml LEF;Pred 5 mg/d SLE01 F 65 11 ANA = 1:2560; IgG CL and anti-dsDNA = pos HCQ,LEF, Pred 5 mg/d SLE02 M 31 3 ANA = 1:640; anti-RNP, anti-Sm, andanti-Ro = pos HCQ, Pred 3 mg/d SLE03 M 63 32 ANA = 1:640; anti-dsDNA andIgG anti-CL = pos HCQ, Pred 5 mg/d SLE04 F 37 5 ANA = 1:2560; anti-Ro =pos None SLE05 F 43 15 ANA = 1:160; RF = 36 IU/ml HCQ, MMF SLE06 F 46 8ANA = pos; anti-Ro = pos MMF 2 g/d, Pred 10 mg/d SLE07 M 31 23 ANA =1:160; anti-dsDNA and IgG anti-CL = pos HCQ SLE08 F 47 10 ANA = 1:2560;anti-dsDNA, anti-Ro and anti-La = pos; None RF = 103 IU/ml SLE09 F 37 16ANA = pos; anti-dsDNA, anti-IgM and IgG CL = pos HCQ; Pred 10 mg/d SjS01F 52 1 ANA = 1:2560; RF = 354 IU/ml; anti-Ro and anti-La = pos NoneSjS02 F 65 15 ANA = 1:2560; RF = 22 IU/ml; anti-Ro = pos HCQ SjS03 F 5737 ANA = 1:160; anti-Ro = pos MMF, Pred 40 mg/d SjS04 F 67 22 ANA =1:2560; RF 110 IU/ml, anti-Ro and anti-La = pos HCQ SjS05 F 60 9 ANA =1:2560; RF = 126 IU/ml, anti-Ro = pos HCQ SjS06 F 58 21 ANA = 1:2560;anti-Ro and anti-La = pos None SjS07 F 41 13 ANA = 1:2560; anti-Ro andanti-La = pos HCQ SjS08 F 59 8 ANA = 1:2560; RF = 508 IU/ml; anti-Ro =pos None SjS09 F 42 4 ANA = 1:2560; RF = 110 IU/ml; anti-Ro and anti-La= pos HCQ SjS10 F 58 5 ANA = 1:2560; anti-Ro and anti-La = pos HCQ, Pred3 mg/d SjS11 66 5 ANA = 1:2560; anti-Ro and anti-La = pos None SjS12-46F 76 13 ANA = 1:2560; anti-Ro = pos; RF = 32 IU/ml None BP01 M 72 0.3Anti-BP180 = 84 U/ml; anti-BP230 = 115 U/ml, no clinical Pred 60 mg/ddisease BP02 M 54 1.2 Anti-BP180 = 72 U/ml; anti-BP230 = neg, noclinical disease MMF, Pred 12 mg/d BP03 F 56 2 Anti-BP180 = 51 U/ml;anti-BP230 = neg, no clinical disease Pred 20 mg/d BP04 M 75 4.3Anti-BP180 = 45 U/ml; anti-BP230 = 3, minimal disease None BP05 F 66 1.8Anti-BP180 = 96 U/ml; anti-BP230 = 131, severe disease None BP06 M 770.5 Anti-BP180 = 5 U/ml; anti-BP230 = 95, minimal disease None BP07 F 6717 Anti-BP180 = 46 U/ml; anti-BP230 = neg, trace disease RTX (20 mosearlier) PF01 M 54 8.6 Anti-DSG1 = 134 U/ml; anti-DSG3 = neg, minimaldisease AZA activity PF02 M 55 9.8 Anti-DSG1 = neg; anti-DSG3 = neg,minimal disease activity RTX (30 mos earlier) PF03 M 46 6.6 Anti-DSG1 =neg; anti-DSG3 = neg, minimal disease activity None PF04 M 50 5.3Anti-DSG1 = neg; anti-DSG3 = neg, minimal disease activity Cellcept 3g/d PF05 M 72 2.8 Anti-DSG1 = neg; anti-DSG3 = neg, no clinical diseaseDapsone 100 mg/d PV01 M 47 2.6 Anti-DSG1 = 24 U/ml; anti-DSG3 = 5171U/ml, mild disease AZA PV02 M 43 3 Anti-DSG1 = neg; anti-DSG3 = 213U/ml, mild disease MMF, Pred 20 mg/d PV03 M 73 3.3 Anti-DSG1 = neg;anti-DSG3 = 948 U/ml, mild disease Pred 12 mg/d PV04 F 55 4.7 Anti-DSG1= neg; anti-DSG3 = 406 U/ml, mild disease RTX (15 mos earlier) PV05 M 598.3 Anti-DSG1 = neg; anti-DSG3 = 50 U/ml, no disease activity AZA PV06 F48 8.6 Anti-DSG1 = neg; anti-DSG3 = 25 U/ml, no disease activity NonePV07 M 45 0.25 Anti-DSG1 = 968; anti-DSG3 = 735, severe disease, ~20%MMF; Pred 80 mg/d body surface area involving ulcerations and erosionsPV08 M 84 0.2 Anti-DSG1 = 75; anti-DSG3 = 146 U/ml, moderate diseaseNone PV09 M 64 9.8 Anti-DSG1 = 14; anti-DSG3 = 115 U/ml, minimal diseasePred 20 mg/d; IFX activity PV10 M 59 3.8 Anti-DSG1 = 1; anti-DSG3 = 49U/ml, no disease activity AZA PV11 M 55 6 Anti-DSG1 = 34; anti-DSG3 = 35U/ml, minimal disease Pred 20 mg/d; MMF 1000 activity mg/d PV12 F 58 9.5Anti-DSG1 = 1; anti-DSG3 = 43 U/ml, trace disease AZA MS01 F 72 54 SPMS,EDSS 6.5, not clinically active None MS02 M 62 24 RRMS, EDSS 6.5,clinically active BIFN MS03 M 33 2 RRMS, EDSS 1.0, disease notclinically active BIFN MS04 M 75 29 SPMS, EDSS 8.0, disease notclinically active ITMTX MS05 M 52 24 PPMS, EDSS 6.5, disease clinicallyactive MMF, pulse steroids MS06 M 55 25 PPMS, EDSS 7.5, diseaseclinically active ITMTX MS07 F 39 16 SPMS, EDSS 7.0, disease notclinically active Natalizumab (2 mos prior) MS08 F 51 7 SPMS, EDSS 5.5,disease not clinically active BIFN ¹Abbreviations: ANA, antinuclear Ab;ADA, adalimumab; AZA, azathioprine; BIFN, beta interferon; BP, bullouspemphigoid; CCP, cyclic citrullinated peptide; CL, cardiolipin; DH,dermatitis herpetiformis; dsDNA, double stranded DNA; DSG, desmoglein;EDSS, disability scale from 0 = normal to 10 = death; ETN, etanercept;HCQ, hydroxychloroquine; IFX, infliximab; ITMTX, intrathecalmethotrexate; LEF, leflunomide; MMF, mycophenolate mofetil; MTX,methotrexate; PF, pemphigus foliaceus; PPMS, primary progressivemultiple sclerosis; Pred, Prednisone; PV, pemphigus vulgaris; RA,rheumatoid arthritis; RF, rheumatoid factor; RRMS, relapsing remittingmultiple sclerosis; RTX, rituximab; SjS, primary Sjögren's syndrome;SLE, lupus; SPMS, secondary progressive multiple sclerosis; SSZ,sulfasalazine; y, year. ² Normal values: anti-BP = 180, anti-BP = 230,anti-DSG1 and anti-DSG3 antibodies < 9 IU/ml.

7.3 Discussion

This example demonstrates the existence of human IL-10-competent B10cells, which were readily identified by their ability to expresscytoplasmic IL-10 after appropriate in vitro stimulation. Peripheralblood B10 cell frequencies were characteristically low (0.6%) in mostindividuals, consistent with their low frequencies in mice. Human B10procells were also identified by their ability to express IL-10 after invitro maturation. Remarkably, the adaptive and innate activationpathways that induced human B10 and B10pro cell generation, maturation,cytoplasmic IL-10 expression, and IL-10 secretion were similar to thoseused by mouse regulatory B10 cells. Human B10 cell frequencies were alsosimilar in the blood, spleen, and lymph nodes (FIG. 2-3 ).

In this example, B10 cells were defined by their IL-10 production usingoptimized stimulation conditions similar to those that have definedmouse B10 and B10pro cells. IL-10-competence remains the best phenotypicmarker for defining human B10 cells. However, freshly isolated blood B10and B10pro cells were also predominantly CD24^(hi)CD27⁺, with ˜60%₀ alsoexpressing CD38 at high levels (FIG. 6A, C). B10 cells were alsopredominantly CD48^(hi) and CD148^(hi) (FIG. 6A). CD148 is considered amarker for human splenic memory B cells (see Tangye et al., 1988, J.Exp. Med 188:1691-1703) and CD48 is upregulated on activated B cells(see Yokoyama et al., 1991, J. Immunol. 146:2192-2200). By contrast,most CD24^(lo)CD27⁻ B cells were not IL-10 competent, even after 48 h ofLPS or CpG stimulation along with agonistic CD40 ligation. CD27expression is a well-characterized marker for memory B cells, althoughsome memory B cells may be CD27⁻ (see Sanz et al., 2008, Sem. Immunol.20:67-82; Klein et al., 1998, J. Exp. Med 188:1679-1689; and Agematsu etal., 2000, Immunol. Today 21:204-206). The CD27⁺ B cell subpopulationcan also expand during the course of autoimmunity and may serve as amarker for disease activity (see Sanz et al., 2008, Sem. Immunol.20:67-82; and Agematsu et al., 2000, Immunol. Today 21:204-206).However, B10 cell frequencies did not parallel the size of the CD27⁺memory B cell pool in the blood of normal donors or in patients withautoimmune diseases, suggesting that these two subsets may be regulatedindependently. Thus, the CD24^(hi)CD27⁺ phenotype of B10 and B10procells indicates that they may either be selected into the memory B cellpool during their development or B10 and B10pro cells represent adistinct B cell subset that shares common cell surface markers withmemory B cells. However, consistent with their memory phenotype, theproliferative capacity of blood B10 cells in response to mitogenstimulation was higher than that for other B cells (FIG. 6F), as is alsoseen in mice (see Yanaba et al., 2009, J. Immunol. 182:7459-7472). Humantransitional B cells are also rare (2-3% of B cells) in adult blood, andare generally CD10⁺CD24^(hi)CD38^(hi) cells that are IgD⁺CD27⁻ (see Simset al., 2005, Blood 105:4390-4398; and Cuss et al., 2006, J. Immunol.176:1506-1516). Given that CD10 expression is a well-accepted marker formost cells within the transitional B cell pool (see Wardemann et al,2003, Science 301:1374-1377), its absence on B10 cells suggests thatthese cells are not recent emigrants from the bone marrow. Thereby,IL-10 competence and elevated proliferative responses characterize thehuman blood B10 cell subset, with most blood B10 cells expressing aCD24^(hi)CD27⁺ phenotype.

While a small subset of blood B cells was inherently competent toexpress IL-10, a subset of blood B cells also had the capacity toacquire IL-10 competence following stimulation with LPS, CpG, and otherTLR agonists (FIGS. 2D and G). Combining TLR stimulation with prolongedCD40 stimulation facilitated the acquisition of IL-10 competence. SinceIL-10 is critical for B cell regulatory activity in mice, the currentstudies demonstrate that B10 cells in normal individuals and autoimmunedisease patients are functionally competent to express IL-10 (FIG.7A-B).

Mean blood B10 cell frequencies inmost patients with SLE, RA, SjS,autoimmune BD, and MS were not significantly different from thoseobserved in normal controls (FIG. 7B). However, four patients did havesignificantly higher B10 or B10⁺B10pro cell frequencies, including twoSLE patients. This also included one PV patient (PV07) with severedisease involving 20% of the skin (Table 1). Significantly increasedB10⁺B10pro cell frequencies were also found in an untreated SLE patient(SLE04). Interestingly, one patient previously treated with rituximabhad elevated B10⁺B10pro cell frequencies (PF02). Thus, some patients mayhave elevated B10/B10pro cell frequencies as found in some autoimmuneprone mouse strains (see Haas et al., 2010, J. Immunol. (in press); andYanaba et al., 2009, J. Immunol. 182:7459-7472) and during inflammation(see Yanaba et al., 2008, Immunity 28:639-650; and Matsushita et al.,2008, J Clin. Invest. 118:3420-3430).

The current assessment of B10 and B10+B10pro cell frequencies inpatients provides a context for previous studies, where spontaneousIL-10 production by resting blood B cells is reported to be dramaticallyhigher in untreated patients with RA, SLE, and systemic sclerosis thanin controls, as measured by RT-PCR and ELISA assays (see Llorente etal., 1994, Arthritis & Rheumatism 37:1647-1655). More recently, B cellcytoplasmic IL-10 production by blood mononuclear cells has beenexamined in patients with SLE compared with normal controls after 24 hin culture with or without PMA plus ionomycin, or LPS (see Amel Kashipazet al., 2003, Lupus 12:356-363). In that study, significantly more SLE Bcells spontaneously produced cytoplasmic IL-10 (1.1%) than controls(0.6%). However, after stimulation by PMA plus ionomycin, the number ofIL-10⁺ B cells was no higher in the SLE patients (1.3%) when comparedwith the unstimulated cultures, but was slightly higher in the controls(1.5%). By contrast, LPS stimulation failed in either case to increaseintracellular IL-10-producing B cell frequencies in comparison withunstimulated cells. Furthermore, unstimulated and stimulated CD5 ⁺ Bcells from SLE patients were enriched for cells producing high levels ofIL-10, although stimulation of CD5 ⁻ B cells from normal controlsinduced more IL-10 production than stimulation of CD5 ⁺ cells fromnormal controls. In other studies, 26% of SLE patients' blood B cellsspontaneously expressed cytoplasmic IL-10, while 50% were IL-10⁺ aftermitogen activation (see Diaz-Alderete et al., 2004, J Autoimmun.23:379-383). In this case, IL-0 expression was confined to a CD154 ⁺(CD40L⁺) subset of B cells, but not to CD5 ⁺ B cells.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described will become apparent to thoseskilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

1-48. (canceled)
 49. A method for treating a disease or conditionassociated with diminished levels of interleukin-10 or ameliorated byincreasing levels of IL-10 comprising administering a therapeuticallyeffective amount of a cellular composition comprising B lymphocyte cellsand a pharmaceutically acceptable carrier, wherein at least 50% of the Blymphocyte cells are characterized as CD24^(high)CD27⁺, to a subject inneed of such treatment, wherein administration of the compositionameliorates the disease or condition.
 50. The method of claim 49,wherein the cells produce IL-10 in the subject.
 51. The method of claim49, in which the disease or condition is inflammation.
 52. The method ofclaim 49, in which the disease or condition is autoimmune disease. 53.The method of claim 49, in which the subject in need of such treatmentis an organ transplant recipient.
 54. The method of claim 49, whereinthe B lymphocyte cells are further characterized by a CD1d^(high)CD5⁺phenotype.
 55. The method of claim 49, wherein the B lymphocyte cellsare further characterized by expression of a marker selected from thegroup consisting of CD19, CD20, CD21, CD22, CD23, CD25, CD38, CD40,CD48, CD72 and CD148.
 56. The method of claim 49, wherein the Blymphocyte cells were contacted ex vivo with a mitogen, a cytokine, agrowth factor, an antibody, a CD40 agonist or a TLR agonist.
 57. Themethod of claim 56, wherein the CD40 agonist is a CD40L (CD154), or anantibody agonist of CD40.
 58. The method of claim 56, wherein the TLRagonist is a TLR1 agonist, a TLR4 agonist, a TLR6 agonist, a TLR7agonist or a TLR9 agonist.
 59. The method of claim 58, wherein theagonist is selected from lipopolysaccharide, CpG oligodeoxynucleotides,Pam3CSK4, Pam2CGDPKHPKSF, or Imiquimod.
 60. The method of claim 49,wherein the B lymphocyte cells were stimulated in vitro or ex vivo withPMA (phorbol 12-myristate 13-acetate) and ionomycin.
 61. The method ofclaim 49, wherein the cellular composition comprises at least 75% B10cells characterized as CD24^(high)CD27⁺.
 62. The method of claim 49,wherein the cellular composition comprises at least 90% B10 cellscharacterized as CD24^(high)CD27⁺.